OMM System Results EC Number Mutation Start End Impact Organism Pubmed ID 1.1.1.1 N249Y 40 533 Asn249Tyr Substitution at the Coenzyme Binding Domain Activates Sulfolobus solfataricus Alcohol Dehydrogenase and Increases Its Thermal Stability† A. Giordano,‡ R. Cannio,‡,§ F. La Cara,‡ S. Bartolucci,| M. Rossi,‡,| and C. A. Raia*,‡ Institute of Protein Biochemistry and Enzymology, CNR, 80125 Naples, Italy, and Department of Organic and Biological Chemistry, UniVersity of Naples “Federico II”, 80134 Naples, Italy ReceiVed September 28, 1998; ReVised Manuscript ReceiVed December 21, 1998 Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 535 770 A mutant of the thermostable NAD+-dependent homotetrameric alcohol dehydrogenase from Sulfolobus solfataricus (SsADH), which has a single substitution, Asn249Tyr, located at the coenzyme binding domain, was obtained by error prone PCR. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 771 1007 The mutant enzyme, which was purified from Escherichia coli to homogeneous form, exhibits a specific activity that is more than 6-fold greater than that of the wild type enzyme, as measured at 65 °C with benzyl alcohol as the substrate. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1266 1399 It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1266 1399 It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1266 1399 It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1400 1652 Spectroscopic studies showed no relevant changes in either secondary or tertiary structure, while analysis with fluorescent probes revealed a significant increase in surface hydrophobicity for the mutant, with respect to that of the wild type molecule. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1653 1815 The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 °C, which was determined by the apparent transition curves. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1653 1815 The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 °C, which was determined by the apparent transition curves. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1653 1815 The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 °C, which was determined by the apparent transition curves. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1816 2043 Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1816 2043 Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 1816 2043 Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2044 2321 Ammonia analysis showed that the Asn249Tyr substitution produced the effect of markedly reducing the extent of deamidation during thermoinactivation, thus suggesting that Asn249 plays a significant role in the mechanism of irreversible thermal denaturation of the archaeal ADH. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2044 2321 Ammonia analysis showed that the Asn249Tyr substitution produced the effect of markedly reducing the extent of deamidation during thermoinactivation, thus suggesting that Asn249 plays a significant role in the mechanism of irreversible thermal denaturation of the archaeal ADH. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2322 2634 Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2322 2634 Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2322 2634 Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 2322 2634 Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 4164 4344 Substitutions at and near the active site in yeast and horse liver alcohol dehydrogenase have been introduced to show how they can affect substrate specificity and reactivity (15). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 4345 4464 More specifically, substitutions at the adenine binding site proved to be able to activate the horse liver enzyme (16). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6355 6602 This paper describes the isolation of an SsADH mutant (Asn249Tyr), which is more active and stable than the wild type enzyme, as shown by the analysis of the kinetic parameters, kinetics of thermal unfolding, and guanidiniumdependent deactivation. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6355 6602 This paper describes the isolation of an SsADH mutant (Asn249Tyr), which is more active and stable than the wild type enzyme, as shown by the analysis of the kinetic parameters, kinetics of thermal unfolding, and guanidiniumdependent deactivation. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6603 6700 Ammonia analysis shows a marked decrease in the deamidation rate upon the Asn249Tyr substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6701 7022 Furthermore, both the decrease in the activating effect by moderate concentrations of denaturants and studies with chelating agents and proteases point to an increase in the structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6701 7022 Furthermore, both the decrease in the activating effect by moderate concentrations of denaturants and studies with chelating agents and proteases point to an increase in the structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6701 7022 Furthermore, both the decrease in the activating effect by moderate concentrations of denaturants and studies with chelating agents and proteases point to an increase in the structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 6701 7022 Furthermore, both the decrease in the activating effect by moderate concentrations of denaturants and studies with chelating agents and proteases point to an increase in the structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 10959 11172 The active pool was dialyzed against buffer A, concentrated as described earlier, and stored frozen at -20 °C. No loss of activity was detected in both wild type and mutant enzyme, after several months of storage. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 12099 12217 Activation and Stabilization in Asn249Tyr SsADH around 340 nm confirmed the result obtained from the emission spectra. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 13821 13924 The mutant enzyme was assayed under the same conditions, but using 30 mM benzyl alcohol and 25 mM NAD+. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 21311 21545 Analysis of the ADH gene sequence isolated from the E. coli clone, which expressed the highest thermophilic and thermoresistant enzyme, revealed only one point mutation (A f T) located at position 1089 of the nucleotide sequence (18). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 21604 21829 Giordano et al. change did not affect the level of gene expression, since optimal conditions for cell growth and induction time were found to be the same for both the recombinant wild type (SsADH) and mutant (mSsADH) enzymes. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 21604 21829 Giordano et al. change did not affect the level of gene expression, since optimal conditions for cell growth and induction time were found to be the same for both the recombinant wild type (SsADH) and mutant (mSsADH) enzymes. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 24148 24325 The rate of the direct and reverse reaction increases as far as an instrumental limit of 93 °C for both the enzymes, although there is a more marked increase for the mutant ADH. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 24148 24325 The rate of the direct and reverse reaction increases as far as an instrumental limit of 93 °C for both the enzymes, although there is a more marked increase for the mutant ADH. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 24705 24752 Activation and Stabilization in Asn249Tyr SsADH Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 27383 27523 The Asn249Tyr replacement considerably changes the catalytic pattern of the wild type enzyme, affecting both its specificity and efficiency. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 27524 27712 The affinity of the mutant decreases for all the alcohols and aldehydes tested, as indicated by Km values, but to a greater extent for the coenzyme (25-fold for NAD+ and 16-fold for NADH). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 27524 27712 The affinity of the mutant decreases for all the alcohols and aldehydes tested, as indicated by Km values, but to a greater extent for the coenzyme (25-fold for NAD+ and 16-fold for NADH). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 28788 28953 However, the emission maximum of the mutant enzyme appears 5 nm redder, and the fluorescence intensity is about 25% higher with respect to that of the wild type ADH. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 29425 29618 Therefore, the fluorescence spectra of SsADH and its mutant were compared at various pHs to detect structural differences in tertiary structure which can be attributed to the additional Tyr249. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 33073 33293 Thermal denaturation of the wild type and mutant ADH was monitored by activity and fluorescence emission at 480 nm after incubation for 30 min at different temperatures and a protein concentration of 10 µg/mL (Figure 3). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 33294 33484 The mutant enzyme seems more thermoresistant than the wild type up to a temperature of 80 °C, after which its activity decreases abruptly, resulting in a transition temperature (3-4 °C) that Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 33294 33484 The mutant enzyme seems more thermoresistant than the wild type up to a temperature of 80 °C, after which its activity decreases abruptly, resulting in a transition temperature (3-4 °C) that Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35104 35151 Activation and Stabilization in Asn249Tyr SsADH Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35153 35471 Biochemistry, Vol. 38, No. 10, 1999 3049 inactivation of both enzymes at a protein concentration of 10 µg/mL in the range of 80-95 °C. The corresponding Arrhenius plots (insets in panels A and B of Figure 4) yield activation energy values of 222 ( 32 and 470 ( 42 kJ/mol for the wild type and mutant ADH, respectively. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35153 35471 Biochemistry, Vol. 38, No. 10, 1999 3049 inactivation of both enzymes at a protein concentration of 10 µg/mL in the range of 80-95 °C. The corresponding Arrhenius plots (insets in panels A and B of Figure 4) yield activation energy values of 222 ( 32 and 470 ( 42 kJ/mol for the wild type and mutant ADH, respectively. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35563 35789 Hq and ∆Sq values for both enzymes do not change with temperature in the range of 8095 °C. ∆∆Gq values between the wild type and mutant ADH are maximal at 80 °C, indicating a higher stability of the latter at this temperature. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35790 36023 In addition, activation entropy is higher for the mutant than for the wild type ADH by about 700 J mol-1 K-1, thus reflecting some loosening of the activated complex or changes in polarity which decrease the level of solvent binding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35790 36023 In addition, activation entropy is higher for the mutant than for the wild type ADH by about 700 J mol-1 K-1, thus reflecting some loosening of the activated complex or changes in polarity which decrease the level of solvent binding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35790 36023 In addition, activation entropy is higher for the mutant than for the wild type ADH by about 700 J mol-1 K-1, thus reflecting some loosening of the activated complex or changes in polarity which decrease the level of solvent binding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 35790 36023 In addition, activation entropy is higher for the mutant than for the wild type ADH by about 700 J mol-1 K-1, thus reflecting some loosening of the activated complex or changes in polarity which decrease the level of solvent binding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36024 36195 The marked increase in Ea and, consequently, in activation enthalpy suggests that some intramolecular stabilizing forces could be introduced by the Asn249Tyr substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36024 36195 The marked increase in Ea and, consequently, in activation enthalpy suggests that some intramolecular stabilizing forces could be introduced by the Asn249Tyr substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36024 36195 The marked increase in Ea and, consequently, in activation enthalpy suggests that some intramolecular stabilizing forces could be introduced by the Asn249Tyr substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36661 36841 The replacement of the Asn249 by a Tyr residue leaves the Asn248 residue to precede the aromatic amino acid which is known to correlate with a relatively low deamidation rate (43). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36842 37104 Therefore, greater ammonia development should be expected to occur from the wild type SsADH, with respect to the mutant enzyme, provided that the thermoinactivation conditions adopted here and structural requirements are favorable to a deamidation reaction (43). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 36842 37104 Therefore, greater ammonia development should be expected to occur from the wild type SsADH, with respect to the mutant enzyme, provided that the thermoinactivation conditions adopted here and structural requirements are favorable to a deamidation reaction (43). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 37335 37393 The mutant enzyme releases less ammonia than the wild type Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 37833 38324 Furthermore, the midpoint of the SsADH thermal deactivation and aggregation transition coincide, whereas the mSsADH aggregation transition precedes the deactivation by about 1 °C. The aggregation is very marked for the mutant ADH at higher protein concentrations, evidently as a consequence of the more extended hydrophobic surface; in fact, at 70 µg/ mL, the aggregation precedes the inactivation of the mutant ADH by about 14 °C, and by only 3 °C that of the wild type (data not reported). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 37833 38324 Furthermore, the midpoint of the SsADH thermal deactivation and aggregation transition coincide, whereas the mSsADH aggregation transition precedes the deactivation by about 1 °C. The aggregation is very marked for the mutant ADH at higher protein concentrations, evidently as a consequence of the more extended hydrophobic surface; in fact, at 70 µg/ mL, the aggregation precedes the inactivation of the mutant ADH by about 14 °C, and by only 3 °C that of the wild type (data not reported). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 40810 40898 enzyme upon exposure to high temperature as a consequence of the Asn249Tyr substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 42382 42539 The c1/2 values, i.e., the denaturant concentration needed to cause 50% inactivation, are 1.7 and 2.2 M GdmCl for the wild type and mutant ADH, respectively. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 42939 43279 The emission spectra of the totally inactive enzymes reveal the disappearance of the single peak at 319 and 324 nm, which is substituted by a band centered at 305-308 nm that is typical of tyrosine emission, and another centered at 351 nm, which is characteristic of tryptophan that is completely exposed to the solvent (data not reported). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 43280 43430 It is important to note that the wild type ADH activity increases at low denaturant concentrations whereas the mutant enzyme is activated very little. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 43280 43430 It is important to note that the wild type ADH activity increases at low denaturant concentrations whereas the mutant enzyme is activated very little. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 43638 43938 DISCUSSION Random mutagenesis by error-prone PCR and a screening method based on heat resistance and thermophilicity tests proved to be a successful strategy for isolating a mutant of the archaeal ADH endowed with increased activity, and improved thermal resistance with respect to the native enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 43638 43938 DISCUSSION Random mutagenesis by error-prone PCR and a screening method based on heat resistance and thermophilicity tests proved to be a successful strategy for isolating a mutant of the archaeal ADH endowed with increased activity, and improved thermal resistance with respect to the native enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 43939 44038 The Asn249Tyr substitution alters the environment of both the coenzyme- and substrate-binding site. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 44570 44652 However, the substrate binding is also substantially affected by the substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 44847 45015 The increased catalytic activity of the mutant enzyme is due to the faster release of the coenzyme as a direct consequence of the major weakening of the binary complex. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 44847 45015 The increased catalytic activity of the mutant enzyme is due to the faster release of the coenzyme as a direct consequence of the major weakening of the binary complex. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 45281 45721 The activation of mSsADH and Cys38-modified SsADH (23) as well as HLADH mutated at the adenosine moiety binding site (16) or modified at essential amino groups (46, 47) suggests a common criterion as a guide for enhancing the turnover of this class of enzymes, namely, to facilitate the coenzyme release by chemical modification or substitutions which are able to electrostatically and/or sterically disturb the coenzyme-enzyme interaction. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 45281 45721 The activation of mSsADH and Cys38-modified SsADH (23) as well as HLADH mutated at the adenosine moiety binding site (16) or modified at essential amino groups (46, 47) suggests a common criterion as a guide for enhancing the turnover of this class of enzymes, namely, to facilitate the coenzyme release by chemical modification or substitutions which are able to electrostatically and/or sterically disturb the coenzyme-enzyme interaction. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 45281 45721 The activation of mSsADH and Cys38-modified SsADH (23) as well as HLADH mutated at the adenosine moiety binding site (16) or modified at essential amino groups (46, 47) suggests a common criterion as a guide for enhancing the turnover of this class of enzymes, namely, to facilitate the coenzyme release by chemical modification or substitutions which are able to electrostatically and/or sterically disturb the coenzyme-enzyme interaction. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 45281 45721 The activation of mSsADH and Cys38-modified SsADH (23) as well as HLADH mutated at the adenosine moiety binding site (16) or modified at essential amino groups (46, 47) suggests a common criterion as a guide for enhancing the turnover of this class of enzymes, namely, to facilitate the coenzyme release by chemical modification or substitutions which are able to electrostatically and/or sterically disturb the coenzyme-enzyme interaction. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47219 47516 SDS-induced equilibrium transition curves for SsADH and mSsADH proved to be similar, with the c1/2 value around 0.13% SDS, after incubation for 60 min at 30 °C. Moderate surfactant concentrations (0.05% SDS) only activated the wild type enzyme (by 115%) but not the mutant enzyme (data not shown). Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47219 47516 SDS-induced equilibrium transition curves for SsADH and mSsADH proved to be similar, with the c1/2 value around 0.13% SDS, after incubation for 60 min at 30 °C. Moderate surfactant concentrations (0.05% SDS) only activated the wild type enzyme (by 115%) but not the mutant enzyme (data not shown). Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47517 47762 Furthermore, SsADH at 70 µg/mL in 0.1 M Tris-HCl (pH 9.0) was activated up to 150%, after incubation for 48 h at 30 °C. However, holo SsADH and holo and apo mutant ADH retained 100% of the initial activity under the same experimental conditions. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47517 47762 Furthermore, SsADH at 70 µg/mL in 0.1 M Tris-HCl (pH 9.0) was activated up to 150%, after incubation for 48 h at 30 °C. However, holo SsADH and holo and apo mutant ADH retained 100% of the initial activity under the same experimental conditions. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47517 47762 Furthermore, SsADH at 70 µg/mL in 0.1 M Tris-HCl (pH 9.0) was activated up to 150%, after incubation for 48 h at 30 °C. However, holo SsADH and holo and apo mutant ADH retained 100% of the initial activity under the same experimental conditions. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47908 48161 The alternative hypothesis was that the mutant molecule was not activated further as it was already endowed with intrinsic flexibility at the catalytic site, and this contrasts with the higher c1/2 value observed for the deactivation of mSsADH by GdmCl. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 47908 48161 The alternative hypothesis was that the mutant molecule was not activated further as it was already endowed with intrinsic flexibility at the catalytic site, and this contrasts with the higher c1/2 value observed for the deactivation of mSsADH by GdmCl. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 48808 48956 The first loses more than 30% of its activity after 3 h at 50 °C, whereas the mutant enzyme retains 100% of its activity even after longer exposure. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 48808 48956 The first loses more than 30% of its activity after 3 h at 50 °C, whereas the mutant enzyme retains 100% of its activity even after longer exposure. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 48808 48956 The first loses more than 30% of its activity after 3 h at 50 °C, whereas the mutant enzyme retains 100% of its activity even after longer exposure. Sulfolobus solfataricus 10074357 1.1.1.1 N248Y 49329 49540 Biochemistry, Vol. 38, No. 10, 1999 NADH quench the fluorescence emission of the mutant SsADH without any shift and energy transfer, suggesting that only small conformational changes affect the protein emission. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 49329 49540 Biochemistry, Vol. 38, No. 10, 1999 NADH quench the fluorescence emission of the mutant SsADH without any shift and energy transfer, suggesting that only small conformational changes affect the protein emission. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 49329 49540 Biochemistry, Vol. 38, No. 10, 1999 NADH quench the fluorescence emission of the mutant SsADH without any shift and energy transfer, suggesting that only small conformational changes affect the protein emission. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 50265 50411 On the other hand, the mutant enzyme binds the fluorescent probe, ANS, very strongly presumably at the same sites present on the wild type enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 50412 50660 A decrease in solvent polarity and, to some extent, an increase in rigidity of the local environment have been demonstrated to increase the quantum yield and decrease the wavelength of emission of amino-substituted naphthalene derivatives (41, 48). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 50412 50660 A decrease in solvent polarity and, to some extent, an increase in rigidity of the local environment have been demonstrated to increase the quantum yield and decrease the wavelength of emission of amino-substituted naphthalene derivatives (41, 48). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 50412 50660 A decrease in solvent polarity and, to some extent, an increase in rigidity of the local environment have been demonstrated to increase the quantum yield and decrease the wavelength of emission of amino-substituted naphthalene derivatives (41, 48). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 50412 50660 A decrease in solvent polarity and, to some extent, an increase in rigidity of the local environment have been demonstrated to increase the quantum yield and decrease the wavelength of emission of amino-substituted naphthalene derivatives (41, 48). Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 51016 51315 The substitution at the coenzyme binding domain of the Asn249 with a tyrosine residue has affected not only the catalysis mechanism but also the structural stability of the archaeal ADH, improving its resistance to heat, denaturants, and proteases, without dramatic changes in the overall structure. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 51016 51315 The substitution at the coenzyme binding domain of the Asn249 with a tyrosine residue has affected not only the catalysis mechanism but also the structural stability of the archaeal ADH, improving its resistance to heat, denaturants, and proteases, without dramatic changes in the overall structure. Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 51469 51811 However, it is evident that the Asn249 plays an important role in the mechanism of irreversible thermal denaturation of SsADH, and that its substitution with a non-heat degradation susceptible residue has produced not only the effect of reducing the deamidation process but also the effect of increasing the rigidity of the molecular edifice. Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 51469 51811 However, it is evident that the Asn249 plays an important role in the mechanism of irreversible thermal denaturation of SsADH, and that its substitution with a non-heat degradation susceptible residue has produced not only the effect of reducing the deamidation process but also the effect of increasing the rigidity of the molecular edifice. Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 51469 51811 However, it is evident that the Asn249 plays an important role in the mechanism of irreversible thermal denaturation of SsADH, and that its substitution with a non-heat degradation susceptible residue has produced not only the effect of reducing the deamidation process but also the effect of increasing the rigidity of the molecular edifice. Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 52124 52319 For example, the role of Asp80Tyr substitution in stabilizing kanamicin nucleotidyltransferase has been found to be correlated with the hydrophobicity of the residue in the protein interior (53). Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 53197 53493 The coincidence of the fluorescence transitions for both the ADHs and the shift of the guanidine-dependent deactivation for the mutant enzyme to higher denaturant concentrations (Figure 6) suggest some stabilization related to catalysis, and this most likely involves the coenzyme binding domain. Sulfolobus solfataricus 10074357 1.1.1.1 D80Y 53197 53493 The coincidence of the fluorescence transitions for both the ADHs and the shift of the guanidine-dependent deactivation for the mutant enzyme to higher denaturant concentrations (Figure 6) suggest some stabilization related to catalysis, and this most likely involves the coenzyme binding domain. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 53647 53976 Proteolysis experiments highlight two important features, namely, the highly limited susceptibility of SsADH to proteolysis, which reflects the considerable structural rigidity achieved by this archaeal protein for functioning at elevated temperatures, and the further tightening of the overall structure induced by the mutation. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 54086 54248 Therefore, it is the decreased flexibility rather than the changes in loop exposure which determines the increased protease resistance shown by the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 54086 54248 Therefore, it is the decreased flexibility rather than the changes in loop exposure which determines the increased protease resistance shown by the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 55579 55801 The Glu97Cys mutant proved to be equally active but less thermostable than native SsADH, showing that at least part of the SsADH thermostability is due to the presence of the glutamate in its structural metal binding site. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 55579 55801 The Glu97Cys mutant proved to be equally active but less thermostable than native SsADH, showing that at least part of the SsADH thermostability is due to the presence of the glutamate in its structural metal binding site. Sulfolobus solfataricus 10074357 1.1.1.1 E97C 55802 55957 It is evident that the replacement of Cys with Glu represents a significant achievement in the evolutionary adaptation of SsADH to thermophilic conditions. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 56340 56635 Activation and Stabilization in Asn249Tyr SsADH As far as the influence of Asn249Tyr substitution on the zinc tightening is concerned, we hypothesize that long-range effects are involved, presumably across hydrogen bond networks or electrostatically through space within the protein scaffolding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 56340 56635 Activation and Stabilization in Asn249Tyr SsADH As far as the influence of Asn249Tyr substitution on the zinc tightening is concerned, we hypothesize that long-range effects are involved, presumably across hydrogen bond networks or electrostatically through space within the protein scaffolding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 56898 57218 Therefore, it is not unreasonable to assume that the strengthening of structural zinc binding in mSsADH is mainly related to hydrogen bond networks involving the metal ligands, and that such interactions contribute greatly to the increased activation enthalpy associated with the thermoinactivation of the mutant enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 57219 57414 This large increase in enthalpy is, however, compensated by an entropy gain, thus resulting in a free energy value which is somewhat higher in the mutant compared to that in the wild type enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 57219 57414 This large increase in enthalpy is, however, compensated by an entropy gain, thus resulting in a free energy value which is somewhat higher in the mutant compared to that in the wild type enzyme. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 57415 57577 In this context, it is evident that the mutation has increased the kinetic thermal stability of SsADH by raising the activation barrier against thermal unfolding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 57415 57577 In this context, it is evident that the mutation has increased the kinetic thermal stability of SsADH by raising the activation barrier against thermal unfolding. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 58703 58967 A single semiconservative substitution at the coenzyme binding domain has shifted this balance slightly toward the former, resulting in greater thermal resistance without any detriment to the specific activity of the enzyme, which in fact proves to be more active. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 58703 58967 A single semiconservative substitution at the coenzyme binding domain has shifted this balance slightly toward the former, resulting in greater thermal resistance without any detriment to the specific activity of the enzyme, which in fact proves to be more active. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 58703 58967 A single semiconservative substitution at the coenzyme binding domain has shifted this balance slightly toward the former, resulting in greater thermal resistance without any detriment to the specific activity of the enzyme, which in fact proves to be more active. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 58968 59115 This study serves as a good example of how it is possible to improve both the stability and activity of a protein, even with the same substitution. Sulfolobus solfataricus 10074357 1.1.1.1 N249Y 58968 59115 This study serves as a good example of how it is possible to improve both the stability and activity of a protein, even with the same substitution. Sulfolobus solfataricus 10074357 1.1.5.2 E277G 749 926 The substitution of Glu277 residue with Gly resulted in a decrease in the K m value for glucose and altered the substrate specificity profile, compared with the wildtype enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 749 926 The substitution of Glu277 residue with Gly resulted in a decrease in the K m value for glucose and altered the substrate specificity profile, compared with the wildtype enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 1082 1317 Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca 2؉, which is responsible for the binding PQQ. Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 1082 1317 Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca 2؉, which is responsible for the binding PQQ. Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 1082 1317 Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca 2؉, which is responsible for the binding PQQ. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277A 1318 1510 A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gln, or to Lys, was constructed and they all showed decreased K m values and altered substrate specificity profiles. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277A 1318 1510 A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gln, or to Lys, was constructed and they all showed decreased K m values and altered substrate specificity profiles. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 1511 1682 Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 1511 1682 Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 L317S 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 K264E 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 T71A 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 K455I 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 L317S 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 R407L 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 S415G 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 T331A 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 K264E 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 T71A 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 K455I 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 R407L 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 S415G 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 T331A 3757 3896 The No.87 mutant showed a decreased K m value and also decreased EDTA tolerance and thermal stability compared with those of the wild type. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 3897 4134 Among these 8 substitutions, we investigated the substitution of Lys264 for Glu, Arg407 for Leu and Glu277 for Gly substitutions (the results were not shown), and found that Glu277Gly showed the similar properties to the No. 87 mutation. Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 9445 9552 The random mutant, No. 87 showed significant decrease in the K m value for glucose, compared with wildtype. Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 9647 9877 The mutant showed the altered substrate specificity toward 2-deoxy-D-glucose, mannose, allose, galactose and for xylose had up to a 2 to 3-fold increase in the relative activity against glucose, compared with the wild-type enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 9647 9877 The mutant showed the altered substrate specificity toward 2-deoxy-D-glucose, mannose, allose, galactose and for xylose had up to a 2 to 3-fold increase in the relative activity against glucose, compared with the wild-type enzyme. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 9878 10065 In addition to the alteration of the K m value and substrate specificity, the No. 87 mutation also resulted in the decrease in the EDTA tolerance (Fig. 2) and thermal stability (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 9878 10065 In addition to the alteration of the K m value and substrate specificity, the No. 87 mutation also resulted in the decrease in the EDTA tolerance (Fig. 2) and thermal stability (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 10245 10352 Glu277Gly also showed a decreased K m value (0.3 mM) and a similar substrate specificity profile as No. 87. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 10245 10352 Glu277Gly also showed a decreased K m value (0.3 mM) and a similar substrate specificity profile as No. 87. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 10353 10443 Glu277Gly also showed a significant decrease in both EDTA tolerance and thermal stability. Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 10588 10802 Among the mutations introduced in the neighboring of Glu277 and Ile278, there was also a decrease in the K m value, but this mutation did not have an impact on the alteration in the substrate specificity (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 10588 10802 Among the mutations introduced in the neighboring of Glu277 and Ile278, there was also a decrease in the K m value, but this mutation did not have an impact on the alteration in the substrate specificity (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 10588 10802 Among the mutations introduced in the neighboring of Glu277 and Ile278, there was also a decrease in the K m value, but this mutation did not have an impact on the alteration in the substrate specificity (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 10588 10802 Among the mutations introduced in the neighboring of Glu277 and Ile278, there was also a decrease in the K m value, but this mutation did not have an impact on the alteration in the substrate specificity (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 10803 10910 Asp275Glu and Asp276Glu resulted in a drastic decrease in the EDTA tolerance (Fig. 2) and a decrease in 822 Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 10803 10910 Asp275Glu and Asp276Glu resulted in a drastic decrease in the EDTA tolerance (Fig. 2) and a decrease in 822 Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 10803 10910 Asp275Glu and Asp276Glu resulted in a drastic decrease in the EDTA tolerance (Fig. 2) and a decrease in 822 Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 10803 10910 Asp275Glu and Asp276Glu resulted in a drastic decrease in the EDTA tolerance (Fig. 2) and a decrease in 822 Acinetobacter calcoaceticus 10544015 1.1.5.2 I278F 12158 12335 The Ile278Phe substitution resulted in an increase in the thermal stability (Table 1) but did not have as much of an effect on EDTA tolerance, as the Asn279His substitution did. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277Q 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277D 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277V 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277A 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277H 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277D 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277N 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277Q 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277H 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277V 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277N 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277A 12524 12673 All the Glu277 variants showed decreased K m values for glucose and altered substrate specificity profile compared with wild-type PQQGDH-B (Table 1). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 12807 13003 Except for Glu277Gly, all of the Glu277 variants showed similar EDTA tolerance to wild-type PQQGDH-B. The thermal stability for Glu277 variants was less than 20% of the wild-type except Glu277Lys. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 12807 13003 Except for Glu277Gly, all of the Glu277 variants showed similar EDTA tolerance to wild-type PQQGDH-B. The thermal stability for Glu277 variants was less than 20% of the wild-type except Glu277Lys. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 13004 13307 Considering that Glu277Lys had a relatively high thermal stability, and in contrast, the other Glu277 variants were very unstable enzymes not suitable for further purification procedures and not practical targets for future application studies, we purified Glu277Lys and analyzed its kinetic parameters. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 13004 13307 Considering that Glu277Lys had a relatively high thermal stability, and in contrast, the other Glu277 variants were very unstable enzymes not suitable for further purification procedures and not practical targets for future application studies, we purified Glu277Lys and analyzed its kinetic parameters. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 13347 13689 The K m value for glucose of purified Glu277Lys was 8.8 mM and was about 40% of that of wild-type PQQGDH-B. The decrease in the K m value was also observed for another substrate, such as for allose (29.0 mM to 21.0 mM), 3-O-methyl-D-glucose (46.0 mM to 27.0 mM), lactose (14.3 mM to 7.5 mM) and for maltose (30.9 mM to 14.3 mM), respectively. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 13766 14048 The wild-type PQQGDH-B showed the highest specific activity for glucose, however, Glu277Lys showed the highest specific activity for allose (5450 U mg Ϫ1), the secondhighest for 3-O-methyl-D-glucose (3820 U mg Ϫ1), and the specific activity for glucose (3668 U mg Ϫ1) was the third. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 13766 14048 The wild-type PQQGDH-B showed the highest specific activity for glucose, however, Glu277Lys showed the highest specific activity for allose (5450 U mg Ϫ1), the secondhighest for 3-O-methyl-D-glucose (3820 U mg Ϫ1), and the specific activity for glucose (3668 U mg Ϫ1) was the third. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 14104 14197 Glu277Lys, 349 s Ϫ1mM Ϫ1, was about three-fold higher than that of wild type (128 s Ϫ1mM Ϫ1). Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 14445 14645 Considering that all the constructed Glu277 variants showed decreased K m values and altered substrate specificity profiles, Glu277 has a crucial role in determining its affinity toward the substrate. Acinetobacter calcoaceticus 10544015 1.1.5.2 D275E 14646 14833 It was also notable that the No. 87 mutant, as well as Glu277Gly, Asp275Glu and Asp276Glu, did not show EDTA tolerance, and were readily inactivated in the presence of 5 mM EDTA (Fig. 2). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277G 14646 14833 It was also notable that the No. 87 mutant, as well as Glu277Gly, Asp275Glu and Asp276Glu, did not show EDTA tolerance, and were readily inactivated in the presence of 5 mM EDTA (Fig. 2). Acinetobacter calcoaceticus 10544015 1.1.5.2 D276E 14646 14833 It was also notable that the No. 87 mutant, as well as Glu277Gly, Asp275Glu and Asp276Glu, did not show EDTA tolerance, and were readily inactivated in the presence of 5 mM EDTA (Fig. 2). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 15033 15223 Since the mutation at Glu277 significantly affected in the catalytic properties of PQQGDH-B, Asp275, Asp276 and Glu277 may construct a putative active site involving the Ca 2ϩ binding sites. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 15224 15361 The kinetic parameters of Glu277Lys showed that this mutation resulted in the increase in the catalytic efficiency of PQQGDH-B (Table 2). Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 15607 15865 As Glu277Lys showed increased specific activity with a low K m value (Table 2), and also retained similar EDTA tolerance and thermal stability as wild-type PQQGDH-B, this variant has great potential in the application for highly-sensitive glucose monitoring. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 15607 15865 As Glu277Lys showed increased specific activity with a low K m value (Table 2), and also retained similar EDTA tolerance and thermal stability as wild-type PQQGDH-B, this variant has great potential in the application for highly-sensitive glucose monitoring. Acinetobacter calcoaceticus 10544015 1.1.5.2 E277K 15607 15865 As Glu277Lys showed increased specific activity with a low K m value (Table 2), and also retained similar EDTA tolerance and thermal stability as wild-type PQQGDH-B, this variant has great potential in the application for highly-sensitive glucose monitoring. Acinetobacter calcoaceticus 10544015 1.1.99.31 R277L 923 1011 The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. Pseudomonas putida 10955993 1.1.99.31 R277K 923 1011 The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. Pseudomonas putida 10955993 1.1.99.31 R277H 923 1011 The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. Pseudomonas putida 10955993 1.1.99.31 R277G 923 1011 The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. Pseudomonas putida 10955993 1.1.99.31 R277K 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277L 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277H 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277G 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277L 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277H 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277G 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277K 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277K 1012 1382 The kcat for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its Km value was 40-fold lower; in contrast, kcats for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and Km values were 5-15-fold lower compared to R277K. The Kds for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. Pseudomonas putida 10955993 1.1.99.31 R277G 1383 1529 The kcat for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued kcat was ∼70% of the wt-MDH value. Pseudomonas putida 10955993 1.1.99.31 R268K 5023 5305 To date, charge-conserved mutations of the equivalent arginine in homologous enzymes (R376K in flavocytochrome b2, R293K in lactate monooxygenase and R268K in lactate oxidase) have resulted in enzymes with extremely low activities, consistent with a critical role of this residue in Pseudomonas putida 10955993 1.1.99.31 R277H 5941 6036 The amounts of the R277 mutant proteins produced upon induction were similar to that of wt-MDH. Pseudomonas putida 10955993 1.1.99.31 R277G 5941 6036 The amounts of the R277 mutant proteins produced upon induction were similar to that of wt-MDH. Pseudomonas putida 10955993 1.1.99.31 R277L 5941 6036 The amounts of the R277 mutant proteins produced upon induction were similar to that of wt-MDH. Pseudomonas putida 10955993 1.1.99.31 R277L 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277H 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277H 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277G 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277G 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277L 6058 6225 The mutant enzymes R277G, R277H, and R277L were observed to be unstable and rapidly inactivated when purified according to the protocol previously used for wt-MDH (9). Pseudomonas putida 10955993 1.1.99.31 R277L 6836 6923 R277G, R277L, and R277H were active and stable when purified by this modified protocol. Pseudomonas putida 10955993 1.1.99.31 R277H 6836 6923 R277G, R277L, and R277H were active and stable when purified by this modified protocol. Pseudomonas putida 10955993 1.1.99.31 R277G 6836 6923 R277G, R277L, and R277H were active and stable when purified by this modified protocol. Pseudomonas putida 10955993 1.1.99.31 R277K 6924 7108 Both wt-MDH and R277K were active when purified in the presence of Triton X-100; however, the stability of both proteins increased significantly when they were purified using Tween 80. Pseudomonas putida 10955993 1.1.99.31 R277K 7109 7313 Additionally, the kcat increased and the Km decreased for R277K. Using the modified protocol, typical yields were low for both wt-MDH and the mutant proteins (∼0.5 mg of protein/g of starting cell paste). Pseudomonas putida 10955993 1.1.99.31 R277K 7109 7313 Additionally, the kcat increased and the Km decreased for R277K. Using the modified protocol, typical yields were low for both wt-MDH and the mutant proteins (∼0.5 mg of protein/g of starting cell paste). Pseudomonas putida 10955993 1.1.99.31 R277K 7109 7313 Additionally, the kcat increased and the Km decreased for R277K. Using the modified protocol, typical yields were low for both wt-MDH and the mutant proteins (∼0.5 mg of protein/g of starting cell paste). Pseudomonas putida 10955993 1.1.99.31 R277K 7109 7313 Additionally, the kcat increased and the Km decreased for R277K. Using the modified protocol, typical yields were low for both wt-MDH and the mutant proteins (∼0.5 mg of protein/g of starting cell paste). Pseudomonas putida 10955993 1.1.99.31 R277K 8369 8646 The pH dependence of the kinetic parameters of R277K as well as of wt-MDH purified according to the modified protocol were measured in a buffer containing 0.052 M ethanolamine, 0.052 M Tris and 0.1 M Mes, using the same equations to fit the data as those described earlier (9). Pseudomonas putida 10955993 1.1.99.31 R277K 8369 8646 The pH dependence of the kinetic parameters of R277K as well as of wt-MDH purified according to the modified protocol were measured in a buffer containing 0.052 M ethanolamine, 0.052 M Tris and 0.1 M Mes, using the same equations to fit the data as those described earlier (9). Pseudomonas putida 10955993 1.1.99.31 R277G 9207 9345 The R277 mutants, especially the ones with neutral side chains, became inactivated to various extents during purification of the proteins. Pseudomonas putida 10955993 1.1.99.31 R277L 9207 9345 The R277 mutants, especially the ones with neutral side chains, became inactivated to various extents during purification of the proteins. Pseudomonas putida 10955993 1.1.99.31 R277G 9346 9526 Thus, due to the low activities and the instability of the mutants of this arginine residue, the nature of its contributions to the reaction mechanism has remained unknown to date. Pseudomonas putida 10955993 1.1.99.31 R277L 9346 9526 Thus, due to the low activities and the instability of the mutants of this arginine residue, the nature of its contributions to the reaction mechanism has remained unknown to date. Pseudomonas putida 10955993 1.1.99.31 R277G 10093 10174 Therefore, it is not surprising that mutations of R277 lead to unstable proteins. Pseudomonas putida 10955993 1.1.99.31 R277H 10093 10174 Therefore, it is not surprising that mutations of R277 lead to unstable proteins. Pseudomonas putida 10955993 1.1.99.31 R277K 10093 10174 Therefore, it is not surprising that mutations of R277 lead to unstable proteins. Pseudomonas putida 10955993 1.1.99.31 R277L 10093 10174 Therefore, it is not surprising that mutations of R277 lead to unstable proteins. Pseudomonas putida 10955993 1.1.99.31 R277G 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277H 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277K 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277L 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277G 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277H 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277K 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277L 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277K 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277L 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277H 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277G 10175 10300 The mutants, R277K, R277G, R277L, and R277H were generated in stable and active forms using a modified purification protocol. Pseudomonas putida 10955993 1.1.99.31 R277H 11567 11959 Binding constants for (R)-mandelate and 1-phenylacetate were also measured kinetically in competitive inhibition experiments (Kis) for R277G (in the presence of 1-methylguanidine), WT, and R277K. Binding of 1-phenylacetate to R277G (in the absence of 1-methylguanidine), R277H, and R277L was only measured through spectral titrations due to the low kcats and high Kms of these mutant enzymes. Pseudomonas putida 10955993 1.1.99.31 R277L 11567 11959 Binding constants for (R)-mandelate and 1-phenylacetate were also measured kinetically in competitive inhibition experiments (Kis) for R277G (in the presence of 1-methylguanidine), WT, and R277K. Binding of 1-phenylacetate to R277G (in the absence of 1-methylguanidine), R277H, and R277L was only measured through spectral titrations due to the low kcats and high Kms of these mutant enzymes. Pseudomonas putida 10955993 1.1.99.31 R277H 11567 11959 Binding constants for (R)-mandelate and 1-phenylacetate were also measured kinetically in competitive inhibition experiments (Kis) for R277G (in the presence of 1-methylguanidine), WT, and R277K. Binding of 1-phenylacetate to R277G (in the absence of 1-methylguanidine), R277H, and R277L was only measured through spectral titrations due to the low kcats and high Kms of these mutant enzymes. Pseudomonas putida 10955993 1.1.99.31 R277L 11567 11959 Binding constants for (R)-mandelate and 1-phenylacetate were also measured kinetically in competitive inhibition experiments (Kis) for R277G (in the presence of 1-methylguanidine), WT, and R277K. Binding of 1-phenylacetate to R277G (in the absence of 1-methylguanidine), R277H, and R277L was only measured through spectral titrations due to the low kcats and high Kms of these mutant enzymes. Pseudomonas putida 10955993 1.1.99.31 R277G 11960 12123 Chemical Rescue of the ActiVity of R277G. The low kcat for R277G could be enhanced by the addition of imidazoles or guanidines to the assay buffer, but not amines. Pseudomonas putida 10955993 1.1.99.31 R277G 11960 12123 Chemical Rescue of the ActiVity of R277G. The low kcat for R277G could be enhanced by the addition of imidazoles or guanidines to the assay buffer, but not amines. Pseudomonas putida 10955993 1.1.99.31 R277G 12124 12425 Typically, the ability of a particular chemical reagent to rescue activity was tested in an assay mixture containing 50 mM potassium phosphate, pH 7.5, 1 mg/mL BSA, 1 mM PMS, 120 µM DCPIP, 0.02-100 µM of the R277G protein, 40-60 mM (S)-mandelate, and high concentrations of the potential rescue agent. Pseudomonas putida 10955993 1.1.99.31 R277K 12625 12865 High concentrations of guanidine hydrochloride, up to 0.4 M, did not affect the activity of wt-MDH or R277K. However, guanidine hydrochloride at concentrations >0.1 M was deleterious for R277G, presumably due to denaturation of the protein. Pseudomonas putida 10955993 1.1.99.31 R277K 12625 12865 High concentrations of guanidine hydrochloride, up to 0.4 M, did not affect the activity of wt-MDH or R277K. However, guanidine hydrochloride at concentrations >0.1 M was deleterious for R277G, presumably due to denaturation of the protein. Pseudomonas putida 10955993 1.1.99.31 R277K 14084 14284 In a preliminary study, we reported that the R277K enzyme, when purified in the presence of the detergent Triton X-100, was active; however, its kcat/Km was 1000-fold lower than the wt-MDH value (16). Pseudomonas putida 10955993 1.1.99.31 R277K 14285 14455 An earlier study of the R277K mutant, using nonhistidyl tagged protein and a longer purification protocol, yielded an enzyme with even lower kcat and kcat/Km values (15). Pseudomonas putida 10955993 1.1.99.31 R277K 14285 14455 An earlier study of the R277K mutant, using nonhistidyl tagged protein and a longer purification protocol, yielded an enzyme with even lower kcat and kcat/Km values (15). Pseudomonas putida 10955993 1.1.99.31 R277K 14456 14665 In this work, using a modified purification scheme, we obtained a highly stable and active R277K mutant enzyme with a kcat/Km only 135-fold lower than that of wt-MDH purified using the same protocol (Table 1). Pseudomonas putida 10955993 1.1.99.31 R277K 14456 14665 In this work, using a modified purification scheme, we obtained a highly stable and active R277K mutant enzyme with a kcat/Km only 135-fold lower than that of wt-MDH purified using the same protocol (Table 1). Pseudomonas putida 10955993 1.1.99.31 R277K 14456 14665 In this work, using a modified purification scheme, we obtained a highly stable and active R277K mutant enzyme with a kcat/Km only 135-fold lower than that of wt-MDH purified using the same protocol (Table 1). Pseudomonas putida 10955993 1.1.99.31 R277K 14456 14665 In this work, using a modified purification scheme, we obtained a highly stable and active R277K mutant enzyme with a kcat/Km only 135-fold lower than that of wt-MDH purified using the same protocol (Table 1). Pseudomonas putida 10955993 1.1.99.31 R277L 14771 14921 R277L, R277G, and R277H were rather unstable and were obtained as 1:1 mixtures of apo- and holoproteins when purified in the presence of Triton X-100. Pseudomonas putida 10955993 1.1.99.31 R277H 14771 14921 R277L, R277G, and R277H were rather unstable and were obtained as 1:1 mixtures of apo- and holoproteins when purified in the presence of Triton X-100. Pseudomonas putida 10955993 1.1.99.31 R277G 14771 14921 R277L, R277G, and R277H were rather unstable and were obtained as 1:1 mixtures of apo- and holoproteins when purified in the presence of Triton X-100. Pseudomonas putida 10955993 1.1.99.31 R277G 15051 15219 Since R277G is highly active in the presence of rescue agents, particularly 1-methylguanidine, the loss in activity could be followed during the purification procedure. Pseudomonas putida 10955993 1.1.99.31 R277G 16110 16249 The mutant enzymes purified by this method were stable and active; however, the yields were typically 10% of those obtained previously (9). Pseudomonas putida 10955993 1.1.99.31 R277G 17038 17251 The effect of the rescue agent on the apparent Km for (S)mandelate for R277G was examined by measuring the Km in the presence of 10 and 80 mM 1-methylguanidine hydrochloride in the standard assay buffer at pH 7.5. Pseudomonas putida 10955993 1.1.99.31 R277G 17278 17496 Differences in the binding energies of the enzyme-substrate (ES) complex and the activation energies for kcat/Km and kcat for wt-MDH and the R277 mutant enzymes were calculated using eqs 2, 3, and 4, respectively (20). Pseudomonas putida 10955993 1.1.99.31 R277G 17278 17496 Differences in the binding energies of the enzyme-substrate (ES) complex and the activation energies for kcat/Km and kcat for wt-MDH and the R277 mutant enzymes were calculated using eqs 2, 3, and 4, respectively (20). Pseudomonas putida 10955993 1.1.99.31 R277K 19759 19906 The rate of reactivity toward oxygen was unaffected in all four R277 mutant proteins as measured by hydrogen peroxide formation in aerobic buffers. Pseudomonas putida 10955993 1.1.99.31 R277K 19907 19972 R277K was completely reduced by (S)-mandelate in aerobic buffers. Pseudomonas putida 10955993 1.1.99.31 R277L 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277G 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277H 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277H 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277L 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277G 19973 20128 R277G, R277L, and R277H could only be partly reduced under aerobic conditions; further reduction was achieved when the buffer was made partially anaerobic. Pseudomonas putida 10955993 1.1.99.31 R277G 20261 20397 In the presence of 20 mM 1-methylguanidine hydrochloride, a fully reduced spectrum was obtained for R277G even under aerobic conditions. Pseudomonas putida 10955993 1.1.99.31 R277H 20400 20499 Methylguanidine hydrochloride did not affect the spectra of either R277H or R277L (data not shown). Pseudomonas putida 10955993 1.1.99.31 R277L 20400 20499 Methylguanidine hydrochloride did not affect the spectra of either R277H or R277L (data not shown). Pseudomonas putida 10955993 1.1.99.31 R277K 20758 20929 The conservative mutant, R277K, was a fairly competent enzyme, with a kcat that was only 4-fold lower compared to wt-MDH; however, surprisingly, its Km was 40-fold higher. Pseudomonas putida 10955993 1.1.99.31 R277K 20758 20929 The conservative mutant, R277K, was a fairly competent enzyme, with a kcat that was only 4-fold lower compared to wt-MDH; however, surprisingly, its Km was 40-fold higher. Pseudomonas putida 10955993 1.1.99.31 R277K 22033 22337 for wt-MDH to 4.6 ( 0.3 for the R277K mutant at pH 7.5, Dkcat/Km also increased from 3.0 ( 0.3 for wtMDH to 5.1 ( 0.4 for R277K. wt-MDH can utilize 2-hydroxy-3-butynoate, vinylglycolate, 3-phenyllactate, or 2-hydroxyoctanoate as substrates but with significantly higher Kms compared to (S)-mandelate (9). Pseudomonas putida 10955993 1.1.99.31 R277K 22033 22337 for wt-MDH to 4.6 ( 0.3 for the R277K mutant at pH 7.5, Dkcat/Km also increased from 3.0 ( 0.3 for wtMDH to 5.1 ( 0.4 for R277K. wt-MDH can utilize 2-hydroxy-3-butynoate, vinylglycolate, 3-phenyllactate, or 2-hydroxyoctanoate as substrates but with significantly higher Kms compared to (S)-mandelate (9). Pseudomonas putida 10955993 1.1.99.31 R277K 22338 22429 No activity was detected with these hydroxy acids for R277K, probably due to very high Kms. Pseudomonas putida 10955993 1.1.99.31 R277H 22624 22885 When the positively charged residue at position 277 was replaced by a neutral residue (glycine, histidine, or leucine), the kcat decreased 400-1000-fold compared to wt-MDH (at neutral and alkaline pHs for R277H, where it is likely to be unprotonated) (Table 1). Pseudomonas putida 10955993 1.1.99.31 R277K 22886 22958 The Km increased 5-15-fold compared to the value for the R277K mutation. Pseudomonas putida 10955993 1.1.99.31 R277H 22959 23103 The activity obtained with R277H was higher at lower pH (26-fold) and Kd for NADPH (95-fold), but only the N107L mutation significantly decreased kcat value. Homo sapiens 15103634 1.1.1.10 N107L 1945 2096 The two mutations increased Km for the substrate (>26-fold) and Kd for NADPH (95-fold), but only the N107L mutation significantly decreased kcat value. Homo sapiens 15103634 1.1.1.10 P34H 3809 4281 XR belongs to the short-chain dehydrogenase/ reductase (SDR) superfamily8 and shares high-sequence identity (63%) with mouse lung carbonyl reductase (MLCR),9 sperm proteins of human (P34H),10 and hamster Abbreviations: XR, L-xylulose reductase; MLCR, mouse lung carbonyl reductase; SDR, short-chain reductase/dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate; P34H, sperm protein of human; P26h, sperm protein of hamster; HSD, hydroxysteroid dehydrogenase. Homo sapiens 15103634 1.1.1.10 G239R 5465 5608 Human XR and P34H are the same gene products because their sequences are identical except for a single amino acid substitution in P34H (G239R). Homo sapiens 15103634 1.1.1.10 P34H 5465 5608 Human XR and P34H are the same gene products because their sequences are identical except for a single amino acid substitution in P34H (G239R). Homo sapiens 15103634 1.1.1.10 P34H 5465 5608 Human XR and P34H are the same gene products because their sequences are identical except for a single amino acid substitution in P34H (G239R). Homo sapiens 15103634 1.1.1.10 Y149F 22551 22819 Differences in coenzyme binding between the two enzymes include the interaction between the O␥ of Ser185 with the O2PA of the pyrophosphate moiety of NADPH, whereas such an interaction is not present in MLCR because of the substitution of this residue with an aspartic Homo sapiens 15103634 1.1.1.10 S136A 22551 22819 Differences in coenzyme binding between the two enzymes include the interaction between the O␥ of Ser185 with the O2PA of the pyrophosphate moiety of NADPH, whereas such an interaction is not present in MLCR because of the substitution of this residue with an aspartic Homo sapiens 15103634 1.1.1.10 K153M 22551 22819 Differences in coenzyme binding between the two enzymes include the interaction between the O␥ of Ser185 with the O2PA of the pyrophosphate moiety of NADPH, whereas such an interaction is not present in MLCR because of the substitution of this residue with an aspartic Homo sapiens 15103634 1.1.1.10 Y149F 27235 27536 The roles of these three residues are supported by site-directed mutagenesis experiments, in which the mutations of S136A, Y149F, and K153M in rat XR resulted in significant decreases in catalytic activity and where only the K153M mutation is accompanied with large increases in Km values for NADP(H). Homo sapiens 15103634 1.1.1.10 S136A 27235 27536 The roles of these three residues are supported by site-directed mutagenesis experiments, in which the mutations of S136A, Y149F, and K153M in rat XR resulted in significant decreases in catalytic activity and where only the K153M mutation is accompanied with large increases in Km values for NADP(H). Homo sapiens 15103634 1.1.1.10 K153M 27235 27536 The roles of these three residues are supported by site-directed mutagenesis experiments, in which the mutations of S136A, Y149F, and K153M in rat XR resulted in significant decreases in catalytic activity and where only the K153M mutation is accompanied with large increases in Km values for NADP(H). Homo sapiens 15103634 1.1.1.10 K153M 27235 27536 The roles of these three residues are supported by site-directed mutagenesis experiments, in which the mutations of S136A, Y149F, and K153M in rat XR resulted in significant decreases in catalytic activity and where only the K153M mutation is accompanied with large increases in Km values for NADP(H). Homo sapiens 15103634 1.1.1.10 N107D 29093 29300 Although the E. coli cell extract of the N107D mutant showed low enzyme activity (73 mU/mg versus 568 mU/mg for the wild-type enzyme), no activity was detected in the cell extract of the N107L mutant enzyme. Homo sapiens 15103634 1.1.1.10 N107L 29301 29383 Because the N107L mutation was in an inactive form, we purified it to homogeneity. Homo sapiens 15103634 1.1.1.10 N107L 29384 29483 The same mutation has been reported to change bacterial 3␤,17␤-HSD into a completely inactive form. Homo sapiens 15103634 1.1.1.10 N107L 29384 29483 The same mutation has been reported to change bacterial 3␤,17␤-HSD into a completely inactive form. Homo sapiens 15103634 1.1.1.10 N107L 29486 29611 The purified N107L mutant form of human XR showed very low activity, with a moderate increase in Km for diacetyl (Table III). Homo sapiens 15103634 1.1.1.10 N107L 29486 29611 The purified N107L mutant form of human XR showed very low activity, with a moderate increase in Km for diacetyl (Table III). Homo sapiens 15103634 1.1.1.10 N107L 29612 29824 The extent of the decrease in kcat value is similar to those caused by the mutations of the catalytically important Ser136, Tyr149, and Lys153,16 supporting the role of Asn107 in the catalytic tetrad in human XR. Homo sapiens 15103634 1.1.1.10 N107L 29612 29824 The extent of the decrease in kcat value is similar to those caused by the mutations of the catalytically important Ser136, Tyr149, and Lys153,16 supporting the role of Asn107 in the catalytic tetrad in human XR. Homo sapiens 15103634 1.1.1.10 N107D 29825 30103 The purified N107D mutant showed a similar change in Km for the substrate but only a small effect on the kcat value, which shows that a small impairment of the active site configuration does not disrupt the proton relay system of the catalytic tetrad in the catalytic mechanism. Homo sapiens 15103634 1.1.1.10 N107D 29825 30103 The purified N107D mutant showed a similar change in Km for the substrate but only a small effect on the kcat value, which shows that a small impairment of the active site configuration does not disrupt the proton relay system of the catalytic tetrad in the catalytic mechanism. Homo sapiens 15103634 1.1.1.10 N107D 30104 30287 It is of interest that both the N107L and N107D mutations significantly increased the Kd value for NADPH (95-fold), which is a larger increase than in bacterial 3␤,17␤-HSD (fivefold). Homo sapiens 15103634 1.1.1.10 N107L 30104 30287 It is of interest that both the N107L and N107D mutations significantly increased the Kd value for NADPH (95-fold), which is a larger increase than in bacterial 3␤,17␤-HSD (fivefold). Homo sapiens 15103634 1.1.1.10 N107L 30104 30287 It is of interest that both the N107L and N107D mutations significantly increased the Kd value for NADPH (95-fold), which is a larger increase than in bacterial 3␤,17␤-HSD (fivefold). Homo sapiens 15103634 1.1.1.10 N107D 30104 30287 It is of interest that both the N107L and N107D mutations significantly increased the Kd value for NADPH (95-fold), which is a larger increase than in bacterial 3␤,17␤-HSD (fivefold). Homo sapiens 15103634 1.1.1.10 N107D 30711 30952 Because the N107D mutation disrupts only the hydrogen bond between this residue and Val86, the structural effect caused by this mutation may be smaller than that caused by the N107L mutation that introduces a hydrophobic and bulkier residue. Homo sapiens 15103634 1.1.1.10 N107L 30711 30952 Because the N107D mutation disrupts only the hydrogen bond between this residue and Val86, the structural effect caused by this mutation may be smaller than that caused by the N107L mutation that introduces a hydrophobic and bulkier residue. Homo sapiens 15103634 1.1.1.10 N107L 33873 34078 In addition, Asn107 may play a role in maintaining the proper orientation of the side-chain of Tyr149 because both N107L and N107D mutations resulted in moderate increases in the Km for the substrate and a Homo sapiens 15103634 1.1.1.10 N107D 33873 34078 In addition, Asn107 may play a role in maintaining the proper orientation of the side-chain of Tyr149 because both N107L and N107D mutations resulted in moderate increases in the Km for the substrate and a Homo sapiens 15103634 2.2.1.1 H103A 861 944 We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 945 1263 It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca2þ (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg2þ (a cation commonly used in ThDP-dependent enzymes studies). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 1381 1568 Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 1381 1568 Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 1381 1568 Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 4905 5134 Replacement of His103 with alanine in TK lowered the enzyme’s affinity for ThDP in the presence of Mg2þ by about eight times [1]; although according to the X-ray data His103 does not come in direct contact with ThDP [7] (Fig. 1). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 8986 9130 The present study was undertaken to elucidate how TK mutation at His103 affects the enzyme’s interaction with ThDP and its catalytic properties. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 10618 10707 centers (0.86 and 2.2 lM) are significantly smaller than that of H103A mutant TK (18 lM). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 10708 11028 To characterise the kinetic parameters of the individual steps of the holoTK reconstitution from the apoenzyme and coenzyme, the reconstitution kinetics of both WT and H103A TK species were investigated in the presence of Mg2þ at different ThDP concentrations and analysed using the set of equations describing Scheme 2. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 11429 11626 Com1 2 3 parison of the primary dissociation constants (Kd ¼ Kd ¼ Kd , Scheme 2) for the WT and H103A TK species in the presence of Mg2þ shows that in the case of mutant TK they are somewhat lower. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 11736 11825 The directionality of these changes suggests the lowering of ThDPs affinity to mutant TK. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 12467 12604 The affinity of H103A apoTK for ThDP in the presence of Mg2þ is much lower than for the WT enzyme, in agreement with earlier reports [1]. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 13070 13256 In the presence of Ca2þ , the apparent Kd value for the second active center of TK was estimated as 0.33 lM for both WT and H103A TK (based on the Scheme 1 and data presented in Fig. 4). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 14337 14540 Comparison of the data on WT TK and H103A TK shows that the values of all kinetic parameters characterizing the reconstitution of the holoenzyme in the presence of Ca2þ are identical for both TK species. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 14654 14850 This result is in agreement with the X-ray crystallography data and indicates the lack of difference in the structures of WT and H103A holoTK upon their reconstitution in the presence of Ca2þ [1]. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 17697 17964 Replacement of His103 with alanine breaks the network of hydrogen bonds in the 40 -NH2 -area thereby causing the local changes in the protein’s conformation and decreasing in this way the affinity of the H103A TK (compared to WT TK) for ThDP in the presence of Mg2þ . Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 18438 18639 Influence of His103 mutation on the catalytic properties of TK Replacement of His103 with alanine does not deprive TK of its catalytic activity but markedly influences its quantitative characteristics. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 18438 18639 Influence of His103 mutation on the catalytic properties of TK Replacement of His103 with alanine does not deprive TK of its catalytic activity but markedly influences its quantitative characteristics. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 18782 18990 The opposite is true for the one-substrate reaction: the mutation increased the rate sevenfold (compare lines 4 and 2, Fig. 6) before becoming equal to the rate of the two-substrate reaction (line 3, Fig. 6). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 18782 18990 The opposite is true for the one-substrate reaction: the mutation increased the rate sevenfold (compare lines 4 and 2, Fig. 6) before becoming equal to the rate of the two-substrate reaction (line 3, Fig. 6). Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 19538 19865 The observation that the mutation of TK at His103 leads to the increase of the one-substrate reaction rate and decrease of the two-substrate reaction rate, so that they become equal, may be considered as experimental substantiation of the earlier advanced hypothesis [1,2] about the intermediate stabilising function of His103. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 19538 19865 The observation that the mutation of TK at His103 leads to the increase of the one-substrate reaction rate and decrease of the two-substrate reaction rate, so that they become equal, may be considered as experimental substantiation of the earlier advanced hypothesis [1,2] about the intermediate stabilising function of His103. Saccharomyces cerevisiae 15178335 2.2.1.1 H103A 19538 19865 The observation that the mutation of TK at His103 leads to the increase of the one-substrate reaction rate and decrease of the two-substrate reaction rate, so that they become equal, may be considered as experimental substantiation of the earlier advanced hypothesis [1,2] about the intermediate stabilising function of His103. Saccharomyces cerevisiae 15178335 2.1.1.63 C145A 657 954 The interaction of dibromomethane (CH2Br2) and bromomethyl acetate (BrCH2OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145S 1109 1295 The formation of AGT-Cys145S-CH2OAc by BrCH2OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys145S-CH2Br adduct from CH2Br2 was too unstable for such characterization. Homo sapiens 15206895 2.1.1.63 C145S 1442 1570 Survival of cells exposed to CH2Br2 was reduced, and the number of mutants was greatly increased when wild-type AGT was present. Homo sapiens 15206895 2.1.1.63 C145S 1442 1570 Survival of cells exposed to CH2Br2 was reduced, and the number of mutants was greatly increased when wild-type AGT was present. Homo sapiens 15206895 2.1.1.63 C145A 1682 1984 Virtually all of the AGTmediated mutants induced by CH2Br2 in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH2OAc was more than 10-fold less genotoxic than CH2Br2 and the survival of cells exposed to BrCH2OAc was not affected by AGT. Homo sapiens 15206895 2.1.1.63 C145A 1682 1984 Virtually all of the AGTmediated mutants induced by CH2Br2 in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH2OAc was more than 10-fold less genotoxic than CH2Br2 and the survival of cells exposed to BrCH2OAc was not affected by AGT. Homo sapiens 15206895 2.1.1.63 C145A 1682 1984 Virtually all of the AGTmediated mutants induced by CH2Br2 in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH2OAc was more than 10-fold less genotoxic than CH2Br2 and the survival of cells exposed to BrCH2OAc was not affected by AGT. Homo sapiens 15206895 2.1.1.63 C145A 1985 2176 The number of mutations (almost all G:C to A:T transitions) induced by BrCH2OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145A 1985 2176 The number of mutations (almost all G:C to A:T transitions) induced by BrCH2OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145S 2567 2802 Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys145S-CH2-DNA adducts) by which CH2Br2 may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs. Homo sapiens 15206895 2.1.1.63 C145S 2567 2802 Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys145S-CH2-DNA adducts) by which CH2Br2 may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs. Homo sapiens 15206895 2.1.1.63 G160R 5923 6147 The recombinant P140K and G160R mutants were purified in a similar way, but the constructs that were used had an MRGSH(H)6GS- sequence at the N-terminal of the protein instead of the C-terminal (H)6 replacement tag (35, 36). Homo sapiens 15206895 2.1.1.63 P140K 5923 6147 The recombinant P140K and G160R mutants were purified in a similar way, but the constructs that were used had an MRGSH(H)6GS- sequence at the N-terminal of the protein instead of the C-terminal (H)6 replacement tag (35, 36). Homo sapiens 15206895 2.1.1.63 C145A 11502 11695 This intermediate leads to a covalent AGT adduct in DNA, which brings about a decreased survival and an increase in the frequency of G:C to A:T transition and G:C to T:A transversion mutations. Homo sapiens 15206895 2.1.1.63 C145A 11502 11695 This intermediate leads to a covalent AGT adduct in DNA, which brings about a decreased survival and an increase in the frequency of G:C to A:T transition and G:C to T:A transversion mutations. Homo sapiens 15206895 2.1.1.63 C145A 11947 12185 We have examined the reactivity of hAGT with CH2Br2 and a model substrate bromomethyl acetate (BrCH2OAc) and have measured the effects of hAGT and an inactive mutant C145A on survival and mutations in cells exposed to CH2Br2 and BrCH2OAc. Homo sapiens 15206895 2.1.1.63 C145A 12186 12413 In contrast to the increase in the mutagenicity and toxicity of CH2Br2 that occurs in the presence of active hAGT, the mutagenicity and toxicity of BrCH2OAc were reduced by active hAGT and enhanced by the inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145A 12186 12413 In contrast to the increase in the mutagenicity and toxicity of CH2Br2 that occurs in the presence of active hAGT, the mutagenicity and toxicity of BrCH2OAc were reduced by active hAGT and enhanced by the inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145A 12186 12413 In contrast to the increase in the mutagenicity and toxicity of CH2Br2 that occurs in the presence of active hAGT, the mutagenicity and toxicity of BrCH2OAc were reduced by active hAGT and enhanced by the inactive C145A mutant. Homo sapiens 15206895 2.1.1.63 C145A 13582 13764 Mutation frequencies were calculated as the number of his revertants that grew on M9 minimal plates lacking histidine over the 108 survivors on M9 plates supplemented with histidine. Homo sapiens 15206895 2.1.1.63 C145A 15088 15196 TRG8 cells that expressed hAGT or the inactive C145A mutant hAGT or no AGT were compared in their responses. Homo sapiens 15206895 2.1.1.63 C145S 15419 15780 BrCH2OAc was used for comparison with CH2Br2 because S-(1-acetoxymethyl)GSH has been successfully applied as a model for studying the reaction of the short-lived S-(1-bromomethyl)GSH intermediate formed by the interaction of GSH and CH2Br2 with nucleosides and DNA (19), and it was expected that the AGT-Cys145S-CH2Br intermediate would also be highly unstable. Homo sapiens 15206895 2.1.1.63 C145A 15781 15945 The presence of active hAGT increased both the toxicity and the mutagenicity of CH2Br2 in a dose-dependent manner, but the C145A mutant had no effect (Figure 1A,B). Homo sapiens 15206895 2.1.1.63 C145A 15946 16183 There was no increase in toxicity, and only a marginal number of mutations in cells lacking hAGT showed that AGT activity is a key factor in mediating the toxic effects of CH2Br2 in the dose range tested (up to 0.1 mM CH2Br2 for 90 min). Homo sapiens 15206895 2.1.1.63 C145A 16184 16344 CH2Br2 was only slightly less effective than BrCH2CH2Br in reducing the survival of cells expressing hAGT but was significantly less potent in inducing mutants. Homo sapiens 15206895 2.1.1.63 C145A 16184 16344 CH2Br2 was only slightly less effective than BrCH2CH2Br in reducing the survival of cells expressing hAGT but was significantly less potent in inducing mutants. Homo sapiens 15206895 2.1.1.63 C145A 16184 16344 CH2Br2 was only slightly less effective than BrCH2CH2Br in reducing the survival of cells expressing hAGT but was significantly less potent in inducing mutants. Homo sapiens 15206895 2.1.1.63 C145A 17362 17458 survivors, whereas BrCH2CH2Br caused a similar reduction in viability but more than 600 mutants. Homo sapiens 15206895 2.1.1.63 C145A 17680 17768 The presence of the C145A mutant hAGT actually slightly increased the loss of viability. Homo sapiens 15206895 2.1.1.63 C145A 17769 17867 A more pronounced difference was observed among the three cell types in terms of mutant induction. Homo sapiens 15206895 2.1.1.63 C145A 17868 18074 While the levels of His revertants increased as the BrCH2OAc concentration rose in all three strains, BrCH2OAc was markedly more mutagenic in cells expressing the C145A mutant than in the other two strains. Homo sapiens 15206895 2.1.1.63 C145A 19413 19612 Because of the unstable nature of these reaction products, mass analysis was attempted for unreacted hAGT, wild-type hAGT, or hAGT mutants incubated with CH2Br2 or BrCH2OAc without trypsin digestion. Homo sapiens 15206895 2.1.1.63 C145S 22663 22773 Therefore, they have masses larger than the carboxyl terminal His6-tagged wild-type and C145S mutant (35, 36). Homo sapiens 15206895 2.1.1.63 P140K 22774 22860 The P140K mutant had an m/z of 23 085, which shifted to 23 153 upon BrCH2OAc addition. Homo sapiens 15206895 2.1.1.63 G160R 22861 22958 The G160R mutant had an m/z of 23 149, which shifted to 23 230 upon BrCH2OAc addition (Figure 4). Homo sapiens 15206895 2.1.1.63 P140K 23351 23492 In contrast, the wild-type, P140K, and G160R mutants showed similar increases in mass upon BrCH2OAc addition (+71, 67, and 82, respectively). Homo sapiens 15206895 2.1.1.63 G160R 23351 23492 In contrast, the wild-type, P140K, and G160R mutants showed similar increases in mass upon BrCH2OAc addition (+71, 67, and 82, respectively). Homo sapiens 15206895 2.1.1.63 C145S 24884 25027 BrCH2OAc, on the other hand, did not produce any detectable amounts of hAGT-DNA conjugates with either wild-type or Cys145Ser hAGT (Figure 5B). Homo sapiens 15206895 2.1.1.63 C145A 27357 27570 As expected from the results in the histidine reversion assay of Figure 1, the frequency of rifampicin resistant (Rifr) mutants produced by CH2Br2 was increased about 100-fold by the expression of hAGT (Figure 7). Homo sapiens 15206895 2.1.1.63 C145A 27571 27750 Sequence analysis of the mutants (Table 1) indicated that the spectrum of mutations induced by CH2Br2 in cells expressing hAGT was quite different from that in cells lacking hAGT. Homo sapiens 15206895 2.1.1.63 C145A 28170 28317 The exposure of cells to the solvent DMSO did not result in any significant increase in mutations in the rpoB gene irrespective of the hAGT status. Homo sapiens 15206895 2.1.1.63 C145A 28318 28527 The spectra of background mutants in TRG8 cells did not reveal any specific induction of G:C to A:T or G:C to T:A mutations (results not shown) and were similar to those reported for a recent large study (40). Homo sapiens 15206895 2.1.1.63 C145S 31346 31444 The AGT-Cys145S-CH2Br formed in this reaction with CH2Br2 would be expected to be highly unstable. Homo sapiens 15206895 2.1.1.63 C145S 32677 32860 This proposed stalling/idling at guanine residues would account for the very high percentage of mutations in the CH2Br2-treated AGT-expressing cells that occur at G:C pairs (Table 1). Homo sapiens 15206895 2.1.1.63 C145S 33888 34291 BrCH2OAc is very active in reacting with Cys145 of the AGT; the combination of the high reactivity of this Cys residue and that of the BrCH2OAc causes the rapid loss of the ability of AGT to repair DNA, shown in Figure 2C,D. The resulting AGT-Cys145S-CH2OAC is unable to form a covalent DNA adduct after noncovalent binding to DNA (Figure 5B); therefore, no increase in mutagenicity or toxicity results. Homo sapiens 15206895 2.1.1.63 C145S 33888 34291 BrCH2OAc is very active in reacting with Cys145 of the AGT; the combination of the high reactivity of this Cys residue and that of the BrCH2OAc causes the rapid loss of the ability of AGT to repair DNA, shown in Figure 2C,D. The resulting AGT-Cys145S-CH2OAC is unable to form a covalent DNA adduct after noncovalent binding to DNA (Figure 5B); therefore, no increase in mutagenicity or toxicity results. Homo sapiens 15206895 2.1.1.63 C145S 33888 34291 BrCH2OAc is very active in reacting with Cys145 of the AGT; the combination of the high reactivity of this Cys residue and that of the BrCH2OAc causes the rapid loss of the ability of AGT to repair DNA, shown in Figure 2C,D. The resulting AGT-Cys145S-CH2OAC is unable to form a covalent DNA adduct after noncovalent binding to DNA (Figure 5B); therefore, no increase in mutagenicity or toxicity results. Homo sapiens 15206895 2.1.1.63 C145A 35268 35443 The ability of the C145A hAGT mutant to increase the mutagenicity and toxicity of BrCH2OAc is quite similar to the ability of this mutant to enhance the response to MNNG (53). Homo sapiens 15206895 2.1.1.63 C145A 35268 35443 The ability of the C145A hAGT mutant to increase the mutagenicity and toxicity of BrCH2OAc is quite similar to the ability of this mutant to enhance the response to MNNG (53). Homo sapiens 15206895 2.1.1.63 C145A 35268 35443 The ability of the C145A hAGT mutant to increase the mutagenicity and toxicity of BrCH2OAc is quite similar to the ability of this mutant to enhance the response to MNNG (53). Homo sapiens 15206895 2.1.1.63 C145A 35584 35733 We therefore suggest that BrCH2OAc forms O6-(CH2OAc)guanine adducts in DNA and that these are recognized by C145A AGT and protected in a similar way. Homo sapiens 15206895 2.1.1.63 C145A 35734 35829 The magnitude of C145A enhancement on response to BrCH2OAc is greater than that seen with MNNG. Homo sapiens 15206895 2.1.1.63 C145A 35734 35829 The magnitude of C145A enhancement on response to BrCH2OAc is greater than that seen with MNNG. Homo sapiens 15206895 2.1.1.63 C145A 35830 35936 This could be due to a tighter binding of the C145A mutant to O6-(CH2OAc)guanine than to O6-methylguanine. Homo sapiens 15206895 2.1.1.63 C145A 36153 36325 The modest effect of wildtype hAGT in reducing the mutations caused by BrCH2OAc may be due to the fact that BrCH2OAc is such a potent direct inactivator of hAGT (Figure 2). Homo sapiens 15206895 2.1.1.63 C145A 36153 36325 The modest effect of wildtype hAGT in reducing the mutations caused by BrCH2OAc may be due to the fact that BrCH2OAc is such a potent direct inactivator of hAGT (Figure 2). Homo sapiens 15206895 2.1.1.63 C145A 36545 36745 The inactivated AGT-Cys145S-CH2COAc would not be able to protect the O6-(CH2OAc)guanine by NER in the same way as the C145A mutant because AGT alkylated at Cys145 is rapidly degraded in vivo (56, 57). Homo sapiens 15206895 2.1.1.63 C145S 36545 36745 The inactivated AGT-Cys145S-CH2COAc would not be able to protect the O6-(CH2OAc)guanine by NER in the same way as the C145A mutant because AGT alkylated at Cys145 is rapidly degraded in vivo (56, 57). Homo sapiens 15206895 2.1.1.63 C145S 37903 38074 However, the AGT-Cys145S-CH2Br conjugate, derived from CH2Br2, is more reactive than the episulfonium ion, AGT-Cys145S+(CH2CH2), derived from BrCH2CH2Br (Figures 5 and 6). Homo sapiens 15206895 2.1.1.63 C145S 38075 38351 These two factors, lesser reactivity of CH2Br2 with AGT but greater reactivity of the resulting complex, may partially cancel out in the cell where much of the AGT is DNA-bound, but the instability of the AGT-Cys145S-CH2Br may cause decomposition before the reaction with DNA. Homo sapiens 15206895 2.1.1.63 C145S 40121 40268 The relatively low stability of the AGT-Cys145S-CH2Br intermediate (Figure 6) may account for the lack of hAGT-CH2Br2 conjugates in MALDI analysis. Homo sapiens 15206895 2.1.1.63 C145S 40269 40540 On the basis of the model of reaction of the hAGT intermediate with DNA and/or water, this instability would be expected for AGT-Cys145S-CH2Br since it cannot undergo cyclic transformation to an episulfonium ion in a way similar to the AGT-Cys145S-CH2CH2Br generated from Homo sapiens 15206895 2.1.1.63 C145S 41565 41712 The AGTCys145S-CH2Br resulting from the reaction with CH2Br2 is able to react with guanine residues in DNA causing mutations and reducing survival. Homo sapiens 15206895 2.1.1.72 D181A 1144 1310 Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base. Escherichia coli 15276835 2.1.1.72 D181A 1144 1310 Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base. Escherichia coli 15276835 2.1.1.72 D181A 17294 17435 Characterisation of the Dam MTase variants We have determined the changes in AP2 fluorescence during binding of different Dam MTase variants. Escherichia coli 15276835 2.1.1.72 D181A 17624 17831 Only a very small change of AP2 fluorescence was observed with the D181A variant in the presence of AdoMet and AdoHcy indicating that the amino acid exchange strongly reduced base flipping as well (Table 2). Escherichia coli 15276835 2.1.1.72 Y184A 18161 18266 In contrast, exchange of the tyrosine residue (Y184A) led to an increase in the P fluorescence (Table 2). Escherichia coli 15276835 2.1.1.72 Y184A 18427 18559 Different from wild-type Ecodam, addition of AdoMet to the binding buffer did not alleviate the fluorescence increase of the mutant. Escherichia coli 15276835 2.1.1.72 Y184A 18427 18559 Different from wild-type Ecodam, addition of AdoMet to the binding buffer did not alleviate the fluorescence increase of the mutant. Escherichia coli 15276835 2.1.1.72 Y184A 18660 18969 The increase in fluorescence of the Y184A variant can be interpreted in the light of the structure of the TaqI - DNA complex, where the aromatic residue corresponding to Tyr184 forms a stacking interaction with the flipped base after the insertion of the target base into the active site pocket of the enzyme. Escherichia coli 15276835 2.1.1.72 Y184A 22155 22296 In contrast, a continuous increase in fluorescence that was much more prominent than that with wild-type was observed with the Y184A variant. Escherichia coli 15276835 2.1.1.72 Y184A 23142 23429 In agreement with this expectation, the variant shows an enlarged equilibrium fluorescence effect in the steady-state experiments (Table 2) and a continuous increase in fluorescence in the stopped-flow experiments that is not followed by a decrease after the initial increase (Figure 4). Escherichia coli 15276835 2.1.1.72 Y184A 23142 23429 In agreement with this expectation, the variant shows an enlarged equilibrium fluorescence effect in the steady-state experiments (Table 2) and a continuous increase in fluorescence in the stopped-flow experiments that is not followed by a decrease after the initial increase (Figure 4). Escherichia coli 15276835 2.1.1.72 Y184A 23142 23429 In agreement with this expectation, the variant shows an enlarged equilibrium fluorescence effect in the steady-state experiments (Table 2) and a continuous increase in fluorescence in the stopped-flow experiments that is not followed by a decrease after the initial increase (Figure 4). Escherichia coli 15276835 2.1.1.72 S188A 29560 29791 Fluorescence stopped-flow studies of base flipping by the S188A and T190A variants We analyzed the kinetics of DNA binding and base flipping of the S188A and T190A variants by similar experiments using the AP2 substrate (Figure 4). Escherichia coli 15276835 2.1.1.72 T190A 29560 29791 Fluorescence stopped-flow studies of base flipping by the S188A and T190A variants We analyzed the kinetics of DNA binding and base flipping of the S188A and T190A variants by similar experiments using the AP2 substrate (Figure 4). Escherichia coli 15276835 2.1.1.72 S188A 29792 30158 Whereas the time-courses of AP2 binding to the T190A variant were indistinguishable from wild-type, the fluorescence increase was delayed in the case of the S188A variant as illustrated by the observation that the wild-type enzyme and T190A show a maximum of the fluorescence well before one second (around 0.8 second) whereas the maximum of fluorescence is observed Escherichia coli 15276835 2.1.1.72 T190A 31221 31375 The kinetics of base flipping by the T190A variant was fitted by very similar rate constants than obtained for the wildtype enzyme (Figure 4 and Table 3). Escherichia coli 15276835 2.1.1.72 T190A 31376 31503 Similarly, the rates of target base binding (k3) were not affected significantly in any of the variants (Figure 4 and Table 3). Escherichia coli 15276835 2.1.1.72 T190A 31711 31959 In contrast, in the case of the S188A variant the quantitative analysis revealed that the rate constant of base flipping is reduced at least seven- to eightfold in the mutant as compared to the lower limit of k2 of the wildtype and T190A (Table 3). Escherichia coli 15276835 2.1.1.72 S188A 31711 31959 In contrast, in the case of the S188A variant the quantitative analysis revealed that the rate constant of base flipping is reduced at least seven- to eightfold in the mutant as compared to the lower limit of k2 of the wildtype and T190A (Table 3). Escherichia coli 15276835 2.1.1.72 S188A 32085 32198 Extensive simulations confirmed the significance of the difference in k2 between wild-type and the S188A variant. Escherichia coli 15276835 2.1.1.72 S188A 32363 32646 In addition, several fits carried out with different starting conditions reproducibly yielded differences in k2 between wild-type and the S188A variant, demonstrating that the difference in k2 is the major reason to explain the different kinetics observed with wild-type and variant. Escherichia coli 15276835 2.1.1.72 S188A 32363 32646 In addition, several fits carried out with different starting conditions reproducibly yielded differences in k2 between wild-type and the S188A variant, demonstrating that the difference in k2 is the major reason to explain the different kinetics observed with wild-type and variant. Escherichia coli 15276835 2.1.1.72 S188A 32363 32646 In addition, several fits carried out with different starting conditions reproducibly yielded differences in k2 between wild-type and the S188A variant, demonstrating that the difference in k2 is the major reason to explain the different kinetics observed with wild-type and variant. Escherichia coli 15276835 2.1.1.72 S188A 32363 32646 In addition, several fits carried out with different starting conditions reproducibly yielded differences in k2 between wild-type and the S188A variant, demonstrating that the difference in k2 is the major reason to explain the different kinetics observed with wild-type and variant. Escherichia coli 15276835 2.1.1.72 S188A 33136 33215 S188A variant, we observed a seven- to eightfold reduced rate of base flipping. Escherichia coli 15276835 2.1.1.72 S188A 38418 38583 By stopped-flow experiments we have shown that the S188A variant displays a reduced rate of base flipping, suggesting that target base rotation is an active process. Escherichia coli 15276835 2.1.1.72 S188A 43006 43218 Rate constants of base flipping of AP2 and other P-substituted substrates were fitted to the data using Scheme 1, which is the minimal scheme to describe the reaction observed, because non-specific binding of the Escherichia coli 15276835 1.1.99.31 G81A 1141 1282 G81A had a higher specificity for small substrates compared to that of wtMDH, though the affinity for (S)-mandelate was relatively unchanged. Pseudomonas putida 15311930 1.1.99.31 G81A 1141 1282 G81A had a higher specificity for small substrates compared to that of wtMDH, though the affinity for (S)-mandelate was relatively unchanged. Pseudomonas putida 15311930 1.1.99.31 G81A 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81S 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81D 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81V 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81V 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81A 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81S 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81D 1283 1402 The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81A 1527 1815 The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 1527 1815 The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 1527 1815 The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 1527 1815 The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81S 1527 1815 The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81S 4454 4808 Despite these differences in the second half-reaction, enzymes in the three subgroups show a high degree of sequence homology and overall threedimensional structural similarity, as illustrated by structural studies of glycolate oxidase from spinach, flavocytochrome b2 from Saccharomyces cereVisiae, and a soluble chimeric mutant of MDH, MDH-GOX2 (7-10). Pseudomonas putida 15311930 1.1.99.31 G81A 4454 4808 Despite these differences in the second half-reaction, enzymes in the three subgroups show a high degree of sequence homology and overall threedimensional structural similarity, as illustrated by structural studies of glycolate oxidase from spinach, flavocytochrome b2 from Saccharomyces cereVisiae, and a soluble chimeric mutant of MDH, MDH-GOX2 (7-10). Pseudomonas putida 15311930 1.1.99.31 G81S 4454 4808 Despite these differences in the second half-reaction, enzymes in the three subgroups show a high degree of sequence homology and overall threedimensional structural similarity, as illustrated by structural studies of glycolate oxidase from spinach, flavocytochrome b2 from Saccharomyces cereVisiae, and a soluble chimeric mutant of MDH, MDH-GOX2 (7-10). Pseudomonas putida 15311930 1.1.99.31 G81A 4454 4808 Despite these differences in the second half-reaction, enzymes in the three subgroups show a high degree of sequence homology and overall threedimensional structural similarity, as illustrated by structural studies of glycolate oxidase from spinach, flavocytochrome b2 from Saccharomyces cereVisiae, and a soluble chimeric mutant of MDH, MDH-GOX2 (7-10). Pseudomonas putida 15311930 1.1.99.31 G81S 6880 7013 More importantly, the G81A and G81S mutants show a higher oxidase activity; this primarily results from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 6880 7013 More importantly, the G81A and G81S mutants show a higher oxidase activity; this primarily results from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81S 6880 7013 More importantly, the G81A and G81S mutants show a higher oxidase activity; this primarily results from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 6880 7013 More importantly, the G81A and G81S mutants show a higher oxidase activity; this primarily results from a higher affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 11046 11278 For flavocytochrome b2 from S. cereVisiae, the corresponding alanine to glycine mutation resulted in an overall decrease in activity without any change in substrate specificity; oxidase activity was not reported for the mutant (13). Pseudomonas putida 15311930 1.1.99.31 G81A 14182 14313 The reoxidation of reduced wtMDH, G81A, or G81S with oxygen was measured in the stopped-flow spectrophotometer at 20 °C as follows. Pseudomonas putida 15311930 1.1.99.31 G81S 14182 14313 The reoxidation of reduced wtMDH, G81A, or G81S with oxygen was measured in the stopped-flow spectrophotometer at 20 °C as follows. Pseudomonas putida 15311930 1.1.99.31 G81S 16489 16634 The levels of expression of the membrane-bound Gly81 mutants were similar to that of wtMDH, with the exception of G81S, which was slightly lower. Pseudomonas putida 15311930 1.1.99.31 G81S 16489 16634 The levels of expression of the membrane-bound Gly81 mutants were similar to that of wtMDH, with the exception of G81S, which was slightly lower. Pseudomonas putida 15311930 1.1.99.31 G81S 16635 16902 Expression of the cytosolic MDH-GOX2 and MDH-GOX2/G81A were similar to that of G81S. The yields of purified protein per gram of cell paste followed a similar pattern; the yield for G81S, MDH-GOX2, and G81A/ MDH-GOX2 was half that of wtMDH and the other Gly81 mutants. Pseudomonas putida 15311930 1.1.99.31 G81A 16635 16902 Expression of the cytosolic MDH-GOX2 and MDH-GOX2/G81A were similar to that of G81S. The yields of purified protein per gram of cell paste followed a similar pattern; the yield for G81S, MDH-GOX2, and G81A/ MDH-GOX2 was half that of wtMDH and the other Gly81 mutants. Pseudomonas putida 15311930 1.1.99.31 G81A 18708 18793 G81A, G81S, and MDH-GOX2/G81A have similar FMN spectra compared to wtMDH and MDHGOX2. Pseudomonas putida 15311930 1.1.99.31 G81V 18794 18965 However, for G81D and G81V, the absorbance peak at 460 nm is blue-shifted to 456 and 455 nm, respectively; additionally, for G81V, the peak at 378 nm is shifted to 365 nm. Pseudomonas putida 15311930 1.1.99.31 G81V 18794 18965 However, for G81D and G81V, the absorbance peak at 460 nm is blue-shifted to 456 and 455 nm, respectively; additionally, for G81V, the peak at 378 nm is shifted to 365 nm. Pseudomonas putida 15311930 1.1.99.31 G81D 18794 18965 However, for G81D and G81V, the absorbance peak at 460 nm is blue-shifted to 456 and 455 nm, respectively; additionally, for G81V, the peak at 378 nm is shifted to 365 nm. Pseudomonas putida 15311930 1.1.99.31 G81D 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81V 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81D 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81V 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81D 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81V 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81D 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81V 19160 19498 When (S)-mandelate was added under anaerobic conditions, all of the proteins were completely reduced with the exception of G81D and G81V. As shown below, these two mutants have extremely low activities; it is probable that the FMN in these mutants is in an orientation such that substrate oxidation and flavin reduction are not favorable. Pseudomonas putida 15311930 1.1.99.31 G81A 19629 19816 The kcat values for G81A and G81S are similar to the kred values, implying that they are similar to wtMDH; the rate-limiting step in catalysis occurs in the first reductive half-reaction. Pseudomonas putida 15311930 1.1.99.31 G81S 19629 19816 The kcat values for G81A and G81S are similar to the kred values, implying that they are similar to wtMDH; the rate-limiting step in catalysis occurs in the first reductive half-reaction. Pseudomonas putida 15311930 1.1.99.31 G81A 19629 19816 The kcat values for G81A and G81S are similar to the kred values, implying that they are similar to wtMDH; the rate-limiting step in catalysis occurs in the first reductive half-reaction. Pseudomonas putida 15311930 1.1.99.31 G81S 19629 19816 The kcat values for G81A and G81S are similar to the kred values, implying that they are similar to wtMDH; the rate-limiting step in catalysis occurs in the first reductive half-reaction. Pseudomonas putida 15311930 1.1.99.31 G81A 19817 19929 G81A has ∼20-fold lower kcat and kred values relative to wtMDH, while the Kd for the substrate is ∼2fold higher. Pseudomonas putida 15311930 1.1.99.31 G81A 19817 19929 G81A has ∼20-fold lower kcat and kred values relative to wtMDH, while the Kd for the substrate is ∼2fold higher. Pseudomonas putida 15311930 1.1.99.31 G81S 19930 20066 The activity of G81S is much lower; its kcat and kred values are ∼150-fold lower, while the affinity for (S)mandelate is 20-fold weaker. Pseudomonas putida 15311930 1.1.99.31 G81S 19930 20066 The activity of G81S is much lower; its kcat and kred values are ∼150-fold lower, while the affinity for (S)mandelate is 20-fold weaker. Pseudomonas putida 15311930 1.1.99.31 G81S 19930 20066 The activity of G81S is much lower; its kcat and kred values are ∼150-fold lower, while the affinity for (S)mandelate is 20-fold weaker. Pseudomonas putida 15311930 1.1.99.31 G81D 20067 20241 The G81D and G81V mutants have extremely low activities, in accordance with our earlier observation that the FMN in these mutants could not be reduced fully by (S)-mandelate. Pseudomonas putida 15311930 1.1.99.31 G81V 20067 20241 The G81D and G81V mutants have extremely low activities, in accordance with our earlier observation that the FMN in these mutants could not be reduced fully by (S)-mandelate. Pseudomonas putida 15311930 1.1.99.31 G81D 20067 20241 The G81D and G81V mutants have extremely low activities, in accordance with our earlier observation that the FMN in these mutants could not be reduced fully by (S)-mandelate. Pseudomonas putida 15311930 1.1.99.31 G81V 20067 20241 The G81D and G81V mutants have extremely low activities, in accordance with our earlier observation that the FMN in these mutants could not be reduced fully by (S)-mandelate. Pseudomonas putida 15311930 1.1.99.31 G81V 20242 20401 The cytosolic chimeric mutant, MDH-GOX2, is highly active and has steady-state parameters that are similar to those of the membrane-bound wtMDH (Table 1) (10). Pseudomonas putida 15311930 1.1.99.31 G81D 20242 20401 The cytosolic chimeric mutant, MDH-GOX2, is highly active and has steady-state parameters that are similar to those of the membrane-bound wtMDH (Table 1) (10). Pseudomonas putida 15311930 1.1.99.31 G81A 22140 22326 The G81A mutation in MDH-GOX2, G81A/MDH-GOX2, has a 100-fold lower kcat relative to MDH-GOX2 itself but a higher affinity for the substrate, unlike the case with wtMDH and the G81A pair. Pseudomonas putida 15311930 1.1.99.31 G81A 22140 22326 The G81A mutation in MDH-GOX2, G81A/MDH-GOX2, has a 100-fold lower kcat relative to MDH-GOX2 itself but a higher affinity for the substrate, unlike the case with wtMDH and the G81A pair. Pseudomonas putida 15311930 1.1.99.31 G81A 22327 22449 The kcat/Km parameter is 20fold lower for G81A relative to wtMDH and 40-fold lower for G81A/MDH-GOX2 relative to MDH-GOX2. Pseudomonas putida 15311930 1.1.99.31 G81A 22327 22449 The kcat/Km parameter is 20fold lower for G81A relative to wtMDH and 40-fold lower for G81A/MDH-GOX2 relative to MDH-GOX2. Pseudomonas putida 15311930 1.1.99.31 G81A 22450 22704 In other words, introducing the alanine mutation at residue 81 results in a similar decrease of the specificity constant for (S)mandelate for both the membrane-bound wtMDH and the cytosolic MDH-GOX2, though it is slightly more deleterious for the latter. Pseudomonas putida 15311930 1.1.99.31 G81A 22810 22904 In contrast, the G81A, G81S, and G81A/MDH-GOX2 mutants all had a much higher Dkcat of 5.2-5.5. Pseudomonas putida 15311930 1.1.99.31 G81S 22810 22904 In contrast, the G81A, G81S, and G81A/MDH-GOX2 mutants all had a much higher Dkcat of 5.2-5.5. Pseudomonas putida 15311930 1.1.99.31 G81A 23129 23224 When the reductive reaction of G81A was monitored at 4 °C, a similar intermediate was observed. Pseudomonas putida 15311930 1.1.99.31 A81G 23466 23697 Substrate Specificity of wtMDH and G81A. Because the introduction of alanine in place of Gly81 resulted in a decrease of the kcat/Km parameter for (S)-mandelate, we next examined the effect of this substitution on other substrates. Pseudomonas putida 15311930 1.1.99.31 G81A 24033 24054 G81A follows the same Pseudomonas putida 15311930 1.1.99.31 G81A 24134 24380 However, unlike the case with (S)-mandelate, similar kcat values are obtained for G81A and wtMDH when using substrates with smaller side chains; additionally, the Km values for the smaller substrates are in fact, lower for G81A relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 24134 24380 However, unlike the case with (S)-mandelate, similar kcat values are obtained for G81A and wtMDH when using substrates with smaller side chains; additionally, the Km values for the smaller substrates are in fact, lower for G81A relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 24134 24380 However, unlike the case with (S)-mandelate, similar kcat values are obtained for G81A and wtMDH when using substrates with smaller side chains; additionally, the Km values for the smaller substrates are in fact, lower for G81A relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 24134 24380 However, unlike the case with (S)-mandelate, similar kcat values are obtained for G81A and wtMDH when using substrates with smaller side chains; additionally, the Km values for the smaller substrates are in fact, lower for G81A relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 24381 24627 In particular, G81A had higher kcat and kcat/Km values than wtMDH for the 4-carbon substrates, 2-hydroxybutyrate and 2-hydroxy-3-butynoate; G81A is a significantly better enzyme relative to wtMDH when using 2-hydroxy-3-butynoate as the substrate. Pseudomonas putida 15311930 1.1.99.31 G81A 24628 24810 A similar observation was made when comparing MDH-GOX2 with G81A/MDH-GOX2; the specificity toward smaller substrates improved for G81A/MDH-GOX2 relative to MDH-GOX2 (data not shown). Pseudomonas putida 15311930 1.1.99.31 G81A 24628 24810 A similar observation was made when comparing MDH-GOX2 with G81A/MDH-GOX2; the specificity toward smaller substrates improved for G81A/MDH-GOX2 relative to MDH-GOX2 (data not shown). Pseudomonas putida 15311930 1.1.99.31 G81A 24628 24810 A similar observation was made when comparing MDH-GOX2 with G81A/MDH-GOX2; the specificity toward smaller substrates improved for G81A/MDH-GOX2 relative to MDH-GOX2 (data not shown). Pseudomonas putida 15311930 1.1.99.31 G81A 25610 25700 Nevertheless, it is evident that G81A has a higher activity with oxygen relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 25701 25772 The kcat/Km(O2) parameter is ∼8-fold higher for G81A relative to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81S 25773 25807 G81S has a similar value as wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81V 25808 25864 G81D and G81V have extremely low activities with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81D 25808 25864 G81D and G81V have extremely low activities with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81V 25865 26029 G81V appears to have an unusually low Km for oxygen; however, given the very low activities and the large errors, the results with G81V and G81D are not conclusive. Pseudomonas putida 15311930 1.1.99.31 G81V 25865 26029 G81V appears to have an unusually low Km for oxygen; however, given the very low activities and the large errors, the results with G81V and G81D are not conclusive. Pseudomonas putida 15311930 1.1.99.31 G81D 25865 26029 G81V appears to have an unusually low Km for oxygen; however, given the very low activities and the large errors, the results with G81V and G81D are not conclusive. Pseudomonas putida 15311930 1.1.99.31 G81A 26114 26191 However, G81A/MDH-GOX2 has a 10-fold higher kcat/Km(O2) relative to MDH-GOX2. Pseudomonas putida 15311930 1.1.99.31 G81A 26192 26380 Therefore, introducing the G81A mutation in wtMDH and in MDH-GOX2, results in a similar increase in activity with oxygen, though the overall rates with the soluble proteins are rather low. Pseudomonas putida 15311930 1.1.99.31 G81A 26192 26380 Therefore, introducing the G81A mutation in wtMDH and in MDH-GOX2, results in a similar increase in activity with oxygen, though the overall rates with the soluble proteins are rather low. Pseudomonas putida 15311930 1.1.99.31 G81A 27283 27509 For the presteady-state kox values, the enzymes were reduced anerobically with 5-fold excess (S)-mandelate for wtMDH and G81A and 50-fold excess (S)-mandelate for G81S, before initiating the reoxidation with oxygenated buffer. Pseudomonas putida 15311930 1.1.99.31 G81A 27818 27948 Because wtMDH and G81A have relatively low Km values with (S)-mandelate (Table 1), they were reduced with 5-fold excess substrate. Pseudomonas putida 15311930 1.1.99.31 G81A 27818 27948 Because wtMDH and G81A have relatively low Km values with (S)-mandelate (Table 1), they were reduced with 5-fold excess substrate. Pseudomonas putida 15311930 1.1.99.31 G81S 27949 28014 G81S could only be fully reduced with a 50-fold excess substrate. Pseudomonas putida 15311930 1.1.99.31 G81S 28249 28359 The kox/ Kd(O2) parameter is ∼23-fold higher for G81A and ∼8-fold higher for G81S, relative to that for wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 28249 28359 The kox/ Kd(O2) parameter is ∼23-fold higher for G81A and ∼8-fold higher for G81S, relative to that for wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81S 28360 28575 The major difference between the two mutants and wtMDH was the substantially increased affinity for oxygen, such that the Kd(O2) values for the mutants were lower than the atmospheric oxygen concentration (0.27 mM). Pseudomonas putida 15311930 1.1.99.31 G81S 28360 28575 The major difference between the two mutants and wtMDH was the substantially increased affinity for oxygen, such that the Kd(O2) values for the mutants were lower than the atmospheric oxygen concentration (0.27 mM). Pseudomonas putida 15311930 1.1.99.31 G81S 28360 28575 The major difference between the two mutants and wtMDH was the substantially increased affinity for oxygen, such that the Kd(O2) values for the mutants were lower than the atmospheric oxygen concentration (0.27 mM). Pseudomonas putida 15311930 1.1.99.31 G81D 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81V 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81D 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81V 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81A 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81S 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81A 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81S 28576 29052 Because of the low activities and high Km values for (S)-mandelate obtained with the other Gly81 mutants (Table 1), we did not attempt stopped-flow measurements of the oxidative reaction for G81D and G81V. The similarity of the steady-state rates of hydrogen peroxide formation and the rate of reoxidation of reduced wtMDH by oxygen in stopped-flow experiments for wtMDH, G81A, and G81S indicates that hydrogen peroxide is the primary product of reducing oxygen and not water. Pseudomonas putida 15311930 1.1.99.31 G81S 29242 29419 The dissociation constants for the adducts with G81A, G81S, and G81D were measured by titration with increasing amounts of sulfite at 4 °C and pH 7.5, as described earlier (18). Pseudomonas putida 15311930 1.1.99.31 G81D 29242 29419 The dissociation constants for the adducts with G81A, G81S, and G81D were measured by titration with increasing amounts of sulfite at 4 °C and pH 7.5, as described earlier (18). Pseudomonas putida 15311930 1.1.99.31 G81A 29242 29419 The dissociation constants for the adducts with G81A, G81S, and G81D were measured by titration with increasing amounts of sulfite at 4 °C and pH 7.5, as described earlier (18). Pseudomonas putida 15311930 1.1.99.31 G81A 29616 29722 These results indicate that the FMN environment in G81A is relatively unchanged relative to that in wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81S 29723 29871 The higher affinity of sulfite for FMN in G81S probably results from an additional interaction of the adduct with the hydroxyl side chain of serine. Pseudomonas putida 15311930 1.1.99.31 G81D 29872 30079 On the other hand, the lack of adduct formation in G81D shows that a negative charge on the re side of FMN near the N5 position drastically alters the affinity for an adduct with a negatively charged ligand. Pseudomonas putida 15311930 1.1.99.31 G81A 30135 30340 We measured the redox potentials of FMN in wtMDH and the mutants to examine whether changes at Gly81 altered the redox potential of FMN and if these changes could be correlated with the oxidase activities. Pseudomonas putida 15311930 1.1.99.31 G81A 30479 30680 wtMDH, MDH-GOX2, and the G81A mutants were observed to stabilize the anionic form of the FMN semiquinone, which has also been observed for other homologues in this protein family (data not shown) (22). Pseudomonas putida 15311930 1.1.99.31 G81D 30733 30855 On introducing the alanine mutation at Gly81 in both wtMDH and MDH-GOX2, the redox potential becomes ∼30 mV more negative. Pseudomonas putida 15311930 1.1.99.31 G81S 30733 30855 On introducing the alanine mutation at Gly81 in both wtMDH and MDH-GOX2, the redox potential becomes ∼30 mV more negative. Pseudomonas putida 15311930 1.1.99.31 G81V 30733 30855 On introducing the alanine mutation at Gly81 in both wtMDH and MDH-GOX2, the redox potential becomes ∼30 mV more negative. Pseudomonas putida 15311930 1.1.99.31 G81S 30856 30991 For G81S, G81D, and G81V, the redox potential of the FMNox/FMNsq couple is ∼20, 30, and ∼15 mV more negative compared to that of wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81V 30856 30991 For G81S, G81D, and G81V, the redox potential of the FMNox/FMNsq couple is ∼20, 30, and ∼15 mV more negative compared to that of wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81D 30856 30991 For G81S, G81D, and G81V, the redox potential of the FMNox/FMNsq couple is ∼20, 30, and ∼15 mV more negative compared to that of wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81D 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81V 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81D 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81V 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81D 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81V 34572 34804 Though all of the Gly81 mutants were similar to wtMDH in terms of expression and stability, there were some differences in the FMN spectra and redox potential, indicating that the change at Gly81 affects the cofactor in subtle ways. Pseudomonas putida 15311930 1.1.99.31 G81V 34805 34879 The absorbance spectra of G81D and G81V were different from that of wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81D 34805 34879 The absorbance spectra of G81D and G81V were different from that of wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81V 35048 35147 However, G81V shows a bigger change in the FMN absorbance spectrum and also extremely low activity. Pseudomonas putida 15311930 1.1.99.31 G81A 35525 35704 Because the dissociation constant of the adduct in G81A was similar to that in wtMDH, we can conclude that the overall environment around the FMN in G81A was relatively unchanged. Pseudomonas putida 15311930 1.1.99.31 G81A 35525 35704 Because the dissociation constant of the adduct in G81A was similar to that in wtMDH, we can conclude that the overall environment around the FMN in G81A was relatively unchanged. Pseudomonas putida 15311930 1.1.99.31 G81D 35705 35957 However, because there was no adduct formation in G81D at all, the FMN N5 atom is probably quite electronreplete in G81D, and the low activity of G81D may result from the inability of the FMN to accept electrons from the substrate during its oxidation. Pseudomonas putida 15311930 1.1.99.31 G81D 35958 36148 Redox potentials of the FMNox/FMNsq couple in wtMDH are different from the Gly81 mutants, but they do not follow the same pattern as the overall catalytic activity or reactivity with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81D 35958 36148 Redox potentials of the FMNox/FMNsq couple in wtMDH are different from the Gly81 mutants, but they do not follow the same pattern as the overall catalytic activity or reactivity with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81S 36452 36577 Aspartate causes a similar change, whereas the serine and valine replacements perturb the redox potential to a lesser extent. Pseudomonas putida 15311930 1.1.99.31 G81A 36452 36577 Aspartate causes a similar change, whereas the serine and valine replacements perturb the redox potential to a lesser extent. Pseudomonas putida 15311930 1.1.99.31 G81S 36452 36577 Aspartate causes a similar change, whereas the serine and valine replacements perturb the redox potential to a lesser extent. Pseudomonas putida 15311930 1.1.99.31 G81A 36452 36577 Aspartate causes a similar change, whereas the serine and valine replacements perturb the redox potential to a lesser extent. Pseudomonas putida 15311930 1.1.99.31 G81A 37038 37148 G81A and G81S show 18- and ∼130fold lower kcat, respectively; this is true for the rate of FMN reduction also. Pseudomonas putida 15311930 1.1.99.31 G81S 37038 37148 G81A and G81S show 18- and ∼130fold lower kcat, respectively; this is true for the rate of FMN reduction also. Pseudomonas putida 15311930 1.1.99.31 G81A 37038 37148 G81A and G81S show 18- and ∼130fold lower kcat, respectively; this is true for the rate of FMN reduction also. Pseudomonas putida 15311930 1.1.99.31 G81S 37038 37148 G81A and G81S show 18- and ∼130fold lower kcat, respectively; this is true for the rate of FMN reduction also. Pseudomonas putida 15311930 1.1.99.31 G81A 37149 37252 Substrate kinetic isotope effects are dramatically higher for G81A and G81S relative to that for wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81S 37149 37252 Substrate kinetic isotope effects are dramatically higher for G81A and G81S relative to that for wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 37956 38134 Interestingly, the transient intermediate was also observed for G81A, though its extremely low extinction coefficient at its optimal wavelength precluded any meaningful analysis. Pseudomonas putida 15311930 1.1.99.31 G81A 38135 38271 However, it was evident that both the rates of formation and breakdown of the intermediate were slow in G81A compared to those in wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 38272 38464 Together with the observation that the redox potential of the FMNox f FMNsq couple is 30 mV more negative in G81A, these results show that the mutation reduces the electrophilicity of the FMN. Pseudomonas putida 15311930 1.1.99.31 G81A 38272 38464 Together with the observation that the redox potential of the FMNox f FMNsq couple is 30 mV more negative in G81A, these results show that the mutation reduces the electrophilicity of the FMN. Pseudomonas putida 15311930 1.1.99.31 G81S 38802 39065 When our results are compared with those obtained with the corresponding mutations in other members of the MDH family, it is evident that the homologous residue has different effects on the substrate oxidation/FMN reduction halfreaction for the different enzymes. Pseudomonas putida 15311930 1.1.99.31 G81A 38802 39065 When our results are compared with those obtained with the corresponding mutations in other members of the MDH family, it is evident that the homologous residue has different effects on the substrate oxidation/FMN reduction halfreaction for the different enzymes. Pseudomonas putida 15311930 1.1.99.31 G81A 38802 39065 When our results are compared with those obtained with the corresponding mutations in other members of the MDH family, it is evident that the homologous residue has different effects on the substrate oxidation/FMN reduction halfreaction for the different enzymes. Pseudomonas putida 15311930 1.1.99.31 G81S 38802 39065 When our results are compared with those obtained with the corresponding mutations in other members of the MDH family, it is evident that the homologous residue has different effects on the substrate oxidation/FMN reduction halfreaction for the different enzymes. Pseudomonas putida 15311930 1.1.99.31 G81A 38802 39065 When our results are compared with those obtained with the corresponding mutations in other members of the MDH family, it is evident that the homologous residue has different effects on the substrate oxidation/FMN reduction halfreaction for the different enzymes. Pseudomonas putida 15311930 1.1.99.31 G81S 39194 39466 The alanine to glycine modification in flavocytochrome b2 resulted in a slight decrease in kred and a modest decrease in substrate affinity, while the glycine to alanine or serine mutations in lactate monooxygenase had little effect on the first half-reaction (13, 15-16). Pseudomonas putida 15311930 1.1.99.31 G81A 39194 39466 The alanine to glycine modification in flavocytochrome b2 resulted in a slight decrease in kred and a modest decrease in substrate affinity, while the glycine to alanine or serine mutations in lactate monooxygenase had little effect on the first half-reaction (13, 15-16). Pseudomonas putida 15311930 1.1.99.31 G81S 39194 39466 The alanine to glycine modification in flavocytochrome b2 resulted in a slight decrease in kred and a modest decrease in substrate affinity, while the glycine to alanine or serine mutations in lactate monooxygenase had little effect on the first half-reaction (13, 15-16). Pseudomonas putida 15311930 1.1.99.31 G81A 39194 39466 The alanine to glycine modification in flavocytochrome b2 resulted in a slight decrease in kred and a modest decrease in substrate affinity, while the glycine to alanine or serine mutations in lactate monooxygenase had little effect on the first half-reaction (13, 15-16). Pseudomonas putida 15311930 1.1.99.31 G81S 39467 39609 However, in MDH, there was a significant decrease in catalytic activity and the rate of the first half-reaction for the G81A and G81S mutants. Pseudomonas putida 15311930 1.1.99.31 G81A 39467 39609 However, in MDH, there was a significant decrease in catalytic activity and the rate of the first half-reaction for the G81A and G81S mutants. Pseudomonas putida 15311930 1.1.99.31 G81A 40058 40214 The most interesting finding in our studies of the mutation at Gly81 is that the affinity for oxygen improved ∼10-fold when glycine was replaced by alanine. Pseudomonas putida 15311930 1.1.99.31 G81S 40058 40214 The most interesting finding in our studies of the mutation at Gly81 is that the affinity for oxygen improved ∼10-fold when glycine was replaced by alanine. Pseudomonas putida 15311930 1.1.99.31 G81A 40284 40404 This is the first instance where a mutation was observed to increase the overall activity with oxygen in the MDH family. Pseudomonas putida 15311930 1.1.99.31 G81A 40593 40793 In MDH and lactate monooxygenase, the glycine to alanine mutation resulted in opposite effects; the activity with oxygen increased modestly in MDH and was drastically reduced in lactate monooxygenase. Pseudomonas putida 15311930 1.1.99.31 G81A 40593 40793 In MDH and lactate monooxygenase, the glycine to alanine mutation resulted in opposite effects; the activity with oxygen increased modestly in MDH and was drastically reduced in lactate monooxygenase. Pseudomonas putida 15311930 1.1.99.31 G81S 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81A 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81S 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81A 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81S 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81A 40862 41156 Structural data of the G81A mutant (studies in progress) may explain why the affinity for oxygen improves in G81A and G81S. It is likely that a larger side chain at residue 81 causes the loop to move away from FMN, creating more space for the oxygen molecule to bind, and an increased affinity. Pseudomonas putida 15311930 1.1.99.31 G81A 41157 41374 The modest improvement in the rate of the reaction with oxygen for G81A is also likely to be due to a more optimal orientation of the bound oxygen molecule, such that it can form the FMN-4a hydroperoxide intermediate. Pseudomonas putida 15311930 1.1.99.31 G81A 41375 41641 Alternatively, it is possible that the change in the hydrogenbonding network with structural water molecules that we observed in the structure of reduced MDH-GOX2 is somewhat altered in reduced G81A, resulting in a different degree of desolvation of the active site. Pseudomonas putida 15311930 1.1.99.31 G81A 41375 41641 Alternatively, it is possible that the change in the hydrogenbonding network with structural water molecules that we observed in the structure of reduced MDH-GOX2 is somewhat altered in reduced G81A, resulting in a different degree of desolvation of the active site. Pseudomonas putida 15311930 1.1.99.31 G81A 41375 41641 Alternatively, it is possible that the change in the hydrogenbonding network with structural water molecules that we observed in the structure of reduced MDH-GOX2 is somewhat altered in reduced G81A, resulting in a different degree of desolvation of the active site. Pseudomonas putida 15311930 1.1.99.31 G81A 41375 41641 Alternatively, it is possible that the change in the hydrogenbonding network with structural water molecules that we observed in the structure of reduced MDH-GOX2 is somewhat altered in reduced G81A, resulting in a different degree of desolvation of the active site. Pseudomonas putida 15311930 1.1.99.31 G81A 41812 42069 Surprisingly, the activities with oxygen for the different Gly81 mutants were not correlated with the redox potential of the FMNox f FMNsq couple, implying that redox potential differences cannot explain the low reactivity of the dehydrogenases with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 41812 42069 Surprisingly, the activities with oxygen for the different Gly81 mutants were not correlated with the redox potential of the FMNox f FMNsq couple, implying that redox potential differences cannot explain the low reactivity of the dehydrogenases with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 41812 42069 Surprisingly, the activities with oxygen for the different Gly81 mutants were not correlated with the redox potential of the FMNox f FMNsq couple, implying that redox potential differences cannot explain the low reactivity of the dehydrogenases with oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 42726 42864 Though the mutations did not alter the affinity for (S)mandelate, the affinity for smaller substrates increased in G81A compared to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 42726 42864 Though the mutations did not alter the affinity for (S)mandelate, the affinity for smaller substrates increased in G81A compared to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 42726 42864 Though the mutations did not alter the affinity for (S)mandelate, the affinity for smaller substrates increased in G81A compared to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 42865 43073 The membrane-binding segment of wtMDH does not suppress the reaction with oxygen, because a soluble mutant MDH-GOX2, in which this segment was absent, had an even lower activity with oxygen compared to wtMDH. Pseudomonas putida 15311930 1.1.99.31 G81A 43259 43407 The rate of the oxidative half-reaction with oxygen increased for the G81A mutants; this was primarily a result of an increased affinity for oxygen. Pseudomonas putida 15311930 1.1.99.31 G81A 43259 43407 The rate of the oxidative half-reaction with oxygen increased for the G81A mutants; this was primarily a result of an increased affinity for oxygen. Pseudomonas putida 15311930 2.1.1.63 E93A 15285 15446 Furthermore, we investigated the resistance of E93A, which is destabilized by 4.2 kJ mol-1 and unfolds one order of magnitude faster than wild-type Tk-MGMT (11). Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 16415 16559 A comparison of stability between wild-type Tk-MGMT and E93A indicates that the stability of E93A does not differ from that of wildtype Tk-MGMT. Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 17456 17535 On the other hand, t1/2unf for E93A was smaller than that of wild-type Tk-MGMT. Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 17536 17679 The t1/2unf,H2O values for E93A was calculated to be 1.0 ± 0.6 × 105 s, which is one order of magnitude smaller than that of wild-type Tk-MGMT. Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 17680 17926 Although the unfolding rate of AdaC is too fast to be determined using our system (<1 s), we were able to show that the unfolding of wild-type Tk-MGMT and E93A is much slower than that of AdaC. The t1/2unf values reached a plateau above 7 mM SDS. Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 18683 18844 The t1/2unf,H2O of E93A in the plateau region is no different from that of wild-type Tk-MGMT, but t1/2unf is smaller than wild-type Tk-MGMT below 7 mM (Fig. 5B). Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 18683 18844 The t1/2unf,H2O of E93A in the plateau region is no different from that of wild-type Tk-MGMT, but t1/2unf is smaller than wild-type Tk-MGMT below 7 mM (Fig. 5B). Thermococcus kodakaraensis 15625320 2.1.1.63 R50E 18845 19051 Structural Features of Slow-Unfolding Kinetics—TkMGMT has a unique ion-pair network in the protein interior that is spread among three helices and between the two major domains (Arg 50-Glu 93-Arg 139) (11). Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 19206 19401 We investigated the equilibrium and kinetic stability of mutant E93A to understand whether this network affecting the equilibrium and the kinetic stabilities with respect to Vol. 136, No. 4, 2004 Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 19474 19710 There was a significant difference in the kinetics between wild-type Tk-MGMT and E93A. These results suggest that the ion-pair network that affects stability against GdnHCl also contributes to stability against organic solvents and SDS. Thermococcus kodakaraensis 15625320 2.1.1.63 E93A 19474 19710 There was a significant difference in the kinetics between wild-type Tk-MGMT and E93A. These results suggest that the ion-pair network that affects stability against GdnHCl also contributes to stability against organic solvents and SDS. Thermococcus kodakaraensis 15625320 2.1.1.72 R124A 2142 2397 Salmonella mutants that lack Dam activity are attenuated for virulence in mice and confer protection against murine typhoid fever (Dueger et al., 2001; Dueger et al., 2003), and inactivation of Dam attenuates H. influenzae virulence (Watson et al., 2004). Escherichia coli 15882618 2.1.1.72 R124A 2142 2397 Salmonella mutants that lack Dam activity are attenuated for virulence in mice and confer protection against murine typhoid fever (Dueger et al., 2001; Dueger et al., 2003), and inactivation of Dam attenuates H. influenzae virulence (Watson et al., 2004). Escherichia coli 15882618 2.1.1.72 R124A 4035 4266 We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. Escherichia coli 15882618 2.1.1.72 R124A 5972 6192 To this end we studied the effect of site-directed mutations in EcoDam altered at residues that are conserved within the Dam family and are known to be involved in specific interaction with the GATC target site in T4Dam. Escherichia coli 15882618 2.1.1.72 R124A 6262 6404 R124A had an overall reduction in catalytic activity but methylated two noncanonical sequences (GATT and GATG) faster than the canonical GATC. Escherichia coli 15882618 2.1.1.72 R124A 6262 6404 R124A had an overall reduction in catalytic activity but methylated two noncanonical sequences (GATT and GATG) faster than the canonical GATC. Escherichia coli 15882618 2.1.1.72 P134G 6405 6561 In contrast, variants P134A and P134G retained full enzymatic activity on GATC but gained the ability to methylate the noncanonical sequences GACC and GAAC. Escherichia coli 15882618 2.1.1.72 P134A 6405 6561 In contrast, variants P134A and P134G retained full enzymatic activity on GATC but gained the ability to methylate the noncanonical sequences GACC and GAAC. Escherichia coli 15882618 2.1.1.72 L122A 6562 6732 In addition, while the L122A variant had slightly reduced activity, it showed a dramatic increase in specificity due to a loss in ability to methylate noncanonical sites. Escherichia coli 15882618 2.1.1.72 L122A 6562 6732 In addition, while the L122A variant had slightly reduced activity, it showed a dramatic increase in specificity due to a loss in ability to methylate noncanonical sites. Escherichia coli 15882618 2.1.1.72 L122A 6562 6732 In addition, while the L122A variant had slightly reduced activity, it showed a dramatic increase in specificity due to a loss in ability to methylate noncanonical sites. Escherichia coli 15882618 2.1.1.72 K11S 17654 17850 The D171-K11-Y174 interaction is likely to be critical for normal function since a K11S substitution virtually abolishes enzyme activity (V.G. Kossykh, S.L. Schlagman, and S.H., unpublished data). Escherichia coli 15882618 2.1.1.72 K16R 17927 18059 The mutant of the corresponding Lys in M.EcoRV (K16R) showed an altered specificity toward the target base (Roth and Jeltsch, 2001). Escherichia coli 15882618 2.1.1.72 R124A 21288 21512 Biochemical Analysis of EcoDam Variants EcoDam has considerable sequence similarity (25% identity) to T4Dam (Hattman et al., 1985) but has significantly higher sequence conservation with Dam enzymes from pathogenic bacteria. Escherichia coli 15882618 2.1.1.72 Y119A 21288 21512 Biochemical Analysis of EcoDam Variants EcoDam has considerable sequence similarity (25% identity) to T4Dam (Hattman et al., 1985) but has significantly higher sequence conservation with Dam enzymes from pathogenic bacteria. Escherichia coli 15882618 2.1.1.72 R124A 22321 22517 The R124A and Y119A variants were the most strongly affected by the Ala substitution; their catalytic activity was reduced more than 100-fold (Figure 5B, and see T4Dam R116 and F111 in Figure 2B). Escherichia coli 15882618 2.1.1.72 R124A 22321 22517 The R124A and Y119A variants were the most strongly affected by the Ala substitution; their catalytic activity was reduced more than 100-fold (Figure 5B, and see T4Dam R116 and F111 in Figure 2B). Escherichia coli 15882618 2.1.1.72 Y119A 22321 22517 The R124A and Y119A variants were the most strongly affected by the Ala substitution; their catalytic activity was reduced more than 100-fold (Figure 5B, and see T4Dam R116 and F111 in Figure 2B). Escherichia coli 15882618 2.1.1.72 Y119A 22321 22517 The R124A and Y119A variants were the most strongly affected by the Ala substitution; their catalytic activity was reduced more than 100-fold (Figure 5B, and see T4Dam R116 and F111 in Figure 2B). Escherichia coli 15882618 2.1.1.72 N120S 22518 22570 N120A, N120S, and L122A were affected only slightly. Escherichia coli 15882618 2.1.1.72 N120A 22518 22570 N120A, N120S, and L122A were affected only slightly. Escherichia coli 15882618 2.1.1.72 L122A 22518 22570 N120A, N120S, and L122A were affected only slightly. Escherichia coli 15882618 2.1.1.72 Y119A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 K139A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 P134A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 P134G 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 Y119A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 K139A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 P134A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 P134G 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 R124A 22571 22776 DNA binding by the R124A variant was reduced 10-fold (accounting for only one-tenth of the drop in catalytic activity), while binding of Y119A, P134A, P134G, and K139A was reduced 2- to 3-fold (Figure 5C). Escherichia coli 15882618 2.1.1.72 K139A 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 N120S 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 R137A 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 N120A 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 Y138A 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 L122A 22777 22927 The other variants (N120A, N120S, L122A, R137A, Y138A, and K139A) did not display any appreciable difference in DNA binding compared to the wild-type. Escherichia coli 15882618 2.1.1.72 R137A 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 N120S 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 K139A 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 L122A 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 Y138A 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 N120A 23787 23934 The flipped target base is labeled as a shaded X. Point mutations made in the EcoDam are indicated (note the differences in numbering of residues). Escherichia coli 15882618 2.1.1.72 Y138A 25899 26112 EcoDam variants altered at residues that might be involved in the recognition of the first base pair (R137A, Y138A, and K139A) did not exhibit any strong changes in methylation activity or specificity (Figure S2). Escherichia coli 15882618 2.1.1.72 R137A 25899 26112 EcoDam variants altered at residues that might be involved in the recognition of the first base pair (R137A, Y138A, and K139A) did not exhibit any strong changes in methylation activity or specificity (Figure S2). Escherichia coli 15882618 2.1.1.72 K139A 25899 26112 EcoDam variants altered at residues that might be involved in the recognition of the first base pair (R137A, Y138A, and K139A) did not exhibit any strong changes in methylation activity or specificity (Figure S2). Escherichia coli 15882618 2.1.1.72 R124A 26569 26691 In agreement with the T4Dam structure, GATG and GATT sites were methyl- ated by R124A faster than the canonical GATC site. Escherichia coli 15882618 2.1.1.72 R124A 26829 27068 Thus, while R124A has a 100fold-reduced rate of DNA methylation at GATC sites relative to wild-type EcoDam, it methylated GATG and GATT sites 2- to 3-fold faster than GATC and 30- to 40-fold faster than the wt enzyme modified GATG or GATT. Escherichia coli 15882618 2.1.1.72 R124A 26829 27068 Thus, while R124A has a 100fold-reduced rate of DNA methylation at GATC sites relative to wild-type EcoDam, it methylated GATG and GATT sites 2- to 3-fold faster than GATC and 30- to 40-fold faster than the wt enzyme modified GATG or GATT. Escherichia coli 15882618 2.1.1.72 R124A 26829 27068 Thus, while R124A has a 100fold-reduced rate of DNA methylation at GATC sites relative to wild-type EcoDam, it methylated GATG and GATT sites 2- to 3-fold faster than GATC and 30- to 40-fold faster than the wt enzyme modified GATG or GATT. Escherichia coli 15882618 2.1.1.72 R124A 26829 27068 Thus, while R124A has a 100fold-reduced rate of DNA methylation at GATC sites relative to wild-type EcoDam, it methylated GATG and GATT sites 2- to 3-fold faster than GATC and 30- to 40-fold faster than the wt enzyme modified GATG or GATT. Escherichia coli 15882618 2.1.1.72 R124A 27069 27177 Therefore, R124A has lost the discriminatory requirement for a C:G base pair at the fourth position of GATC. Escherichia coli 15882618 2.1.1.72 R124A 27377 27610 A comparison of specificity factors for the recognition of position 4 (S4) reveals that the R124A variant has an 8000-fold-changed relative preference for methylation of near-cognate sites modified at the fourth position (Figure 6F). Escherichia coli 15882618 2.1.1.72 R124A 27663 27833 Furthermore, the R124A variant retained (or even increased) its specificity for the first and third positions in GATC, so this is a base pair-specific change (Figure 6B). Escherichia coli 15882618 2.1.1.72 R124A 27663 27833 Furthermore, the R124A variant retained (or even increased) its specificity for the first and third positions in GATC, so this is a base pair-specific change (Figure 6B). Escherichia coli 15882618 2.1.1.72 P126S 28019 28235 Naturally occurring variant phage enzymes (T2Damh and T4Damh), which efficiently modify GACC sites in addition to the canonical GATC site (Brooks and Hattman, 1978), contain a P126S substitution (Miner et al., 1989). Escherichia coli 15882618 2.1.1.72 L122A 29162 29288 The specificity factor of wild-type EcoDam was 540; the value was increased at least 30-fold in the case of the L122A variant. Escherichia coli 15882618 2.1.1.72 L122A 29289 29448 Because no activity at near-cognate sites could be detected with the L122A variant, the specificity factor given here is a lower limit, indicated by the arrow. Escherichia coli 15882618 2.1.1.72 P134G 29449 29533 The specificities of the R124A, P134A, and P134G variants were dramatically reduced. Escherichia coli 15882618 2.1.1.72 R124A 29449 29533 The specificities of the R124A, P134A, and P134G variants were dramatically reduced. Escherichia coli 15882618 2.1.1.72 P134A 29449 29533 The specificities of the R124A, P134A, and P134G variants were dramatically reduced. Escherichia coli 15882618 2.1.1.72 P134A 29875 30052 However, P134A exhibited a significant increase in methylation rates of GAAC and GACC substrates, with GACC being modified at almost the same rate as canonical GATC (Figure 6C). Escherichia coli 15882618 2.1.1.72 P134A 29875 30052 However, P134A exhibited a significant increase in methylation rates of GAAC and GACC substrates, with GACC being modified at almost the same rate as canonical GATC (Figure 6C). Escherichia coli 15882618 2.1.1.72 L122A 30868 30986 The activity of the L122A variant of EcoDam (M114 in T4Dam; Figures 2B and 2C) is not appreciably reduced (Figure 5B). Escherichia coli 15882618 2.1.1.72 L122A 31096 31187 This indicates that the L122A variant has a significantly improved specificity (Figure 6H). Escherichia coli 15882618 2.1.1.72 L122A 31311 31612 Whereas the L122A change alone does not severely reduce catalytic activity on the normal GATC substrate, a combination of L122A with the change of any of the base pairs in the recognition site may disturb synergistically the protein-DNA interface, and this could explain the complete loss of activity. Escherichia coli 15882618 2.1.1.72 L122A 31311 31612 Whereas the L122A change alone does not severely reduce catalytic activity on the normal GATC substrate, a combination of L122A with the change of any of the base pairs in the recognition site may disturb synergistically the protein-DNA interface, and this could explain the complete loss of activity. Escherichia coli 15882618 2.1.1.72 Y119A 31944 32010 As shown in Figure 5B, Y119A was the second-most-affected variant. Escherichia coli 15882618 2.1.1.72 N120A 32182 32403 In contrast, removal of the side chain of N120 (N120A) had only a minor effect on methylation rate, although the structure of the specific T4Dam-DNA complex suggests an important role for this amino acid in base flipping. Escherichia coli 15882618 2.1.1.72 N136A 36741 36871 This result agrees with a similar conclusion drawn from rapid kinetics experiments with M.EcoRV variants (Beck and Jeltsch, 2002). Escherichia coli 15882618 2.1.1.72 R124A 37837 37960 We analyzed the biochemical effects of altering the contacts described above in double-mutant cycles (Fersht et al., 1992). Escherichia coli 15882618 2.1.1.72 R124A 38074 38219 We found that it was possible to predictably design MTase variants that no longer recognize one specific base pair within their recognition site. Escherichia coli 15882618 2.1.1.72 R124A 38220 38363 The EcoDam R124A variant displayed a change in specificity because it had a significantly higher catalytic activity toward a near-cognate site. Escherichia coli 15882618 2.1.1.72 P134A 38364 38585 In addition, the EcoDam P134A variant (the analog of the T4Damh MTase) methylated a near-cognate site at almost the same rate as wild-type EcoDam modified the canonical site, indicating a broadened specificity (Figure 6). Escherichia coli 15882618 2.1.1.72 P134A 38364 38585 In addition, the EcoDam P134A variant (the analog of the T4Damh MTase) methylated a near-cognate site at almost the same rate as wild-type EcoDam modified the canonical site, indicating a broadened specificity (Figure 6). Escherichia coli 15882618 2.1.1.72 P134A 39570 39693 Disruption of the contact by removal of the amino acid side chain led to a strongly reduced activity of the enzyme variant. Escherichia coli 15882618 2.1.1.72 P134G 39570 39693 Disruption of the contact by removal of the amino acid side chain led to a strongly reduced activity of the enzyme variant. Escherichia coli 15882618 2.1.1.72 P134A 39570 39693 Disruption of the contact by removal of the amino acid side chain led to a strongly reduced activity of the enzyme variant. Escherichia coli 15882618 2.1.1.72 P134G 39570 39693 Disruption of the contact by removal of the amino acid side chain led to a strongly reduced activity of the enzyme variant. Escherichia coli 15882618 2.1.1.72 P134A 40190 40432 This is illustrated by the high activity and broadened specificity of EcoDam variants P134A and P134G. Experimental Procedures Crystallography T4Dam was expressed and purified as described previously (Kossykh et al., 1995; Yang et al., 2003). Escherichia coli 15882618 2.1.1.72 P134G 40190 40432 This is illustrated by the high activity and broadened specificity of EcoDam variants P134A and P134G. Experimental Procedures Crystallography T4Dam was expressed and purified as described previously (Kossykh et al., 1995; Yang et al., 2003). Escherichia coli 15882618 2.1.1.63 C145S 1736 1932 A rapid increase of fluorescence (k ϳ200 s؊1) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Homo sapiens 16000301 2.1.1.63 C145S 2290 2492 The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions. Homo sapiens 16000301 2.1.1.63 R128G 17593 17766 Mutations at Arg128 and Tyr114, two residues implicated from site-directed mutagenesis and crystallography work (5, 22, 23), significantly reduced the AGT repair efficiency. Homo sapiens 16000301 2.1.1.63 R128G 17767 17883 The mutations had smaller effects with the O6-BzG substrate than the O6-MeG, except in the case of R128G (Table II). Homo sapiens 16000301 2.1.1.63 C145S 19244 19455 The rates of dissociation with the O6-alkylmodified DNA could not be measured with catalytically active AGT, and therefore either the C145A and C145S mutants (Table III) or the alkylated AGT (Table IV) was used. Homo sapiens 16000301 2.1.1.63 C145A 19244 19455 The rates of dissociation with the O6-alkylmodified DNA could not be measured with catalytically active AGT, and therefore either the C145A and C145S mutants (Table III) or the alkylated AGT (Table IV) was used. Homo sapiens 16000301 2.1.1.63 C145S 19456 19586 The results with unmodified DNA (Table IV) indicate that mutation or alkylation of Cys145 did not alter the rates of dissociation. Homo sapiens 16000301 2.1.1.63 C145A 19456 19586 The results with unmodified DNA (Table IV) indicate that mutation or alkylation of Cys145 did not alter the rates of dissociation. Homo sapiens 16000301 2.1.1.63 R128L 20856 21033 AGTC145A, mutant totally devoid of activity, was added to a pyrrolo dC DNA duplex, and the fluorescence changes were recorded (using buffer for background subtraction) (Fig. 5). Homo sapiens 16000301 2.1.1.63 C145S 21494 21608 These titration experiments were also done with the AGT-C145S mutant, which generated similar results (not shown). Homo sapiens 16000301 2.1.1.63 R128G 22514 22650 Consistently, when Arg128 was replaced by Gly to abolish the base flipping “arm” (5), there was only a very limited fluorescence change. Homo sapiens 16000301 2.1.1.63 C145S 30154 30308 Terashima et al. (34) considered the removal of several substituted benzyl derivatives and reported no differences among the substituted benzyl compounds. Homo sapiens 16000301 2.1.1.63 C145S 30613 30848 AGT (C145S) was mixed with the oligonucleotide duplex with G or modified G opposite pyrrolo dC, and changes in fluorescence were monitored, using an excitation wavelength of 370 nm and collecting emission Ͼ400 nm with an end-on filter. Homo sapiens 16000301 2.1.1.63 R128G 33399 33512 With R128G, the attenuation of the rate of observable base flipping (Fig. 6) and the dealkylation activity (Figs. Homo sapiens 16000301 2.1.1.63 R128G 33541 33687 Thus the substitution at Arg128 is consistent with a model in which the base flipping rate is reduced ϳ20-fold, but other steps are not perturbed. Homo sapiens 16000301 2.1.1.63 R128G 33541 33687 Thus the substitution at Arg128 is consistent with a model in which the base flipping rate is reduced ϳ20-fold, but other steps are not perturbed. Homo sapiens 16000301 2.1.1.63 R128G 33881 33998 In modeling with this mutant, the lower activity could be attributed to either k3 or k4 with the data sets available. Homo sapiens 16000301 2.1.1.63 R128G 38737 38921 The model can be used to rationalize the effects of AGT mutants proposed to be involved in base flipping and the difference in the rates of repair of O6-MeG and O6-BzG moieties in DNA. Homo sapiens 16000301 1.10.99.2 N161H 1148 1332 Mutation of Asn161 to His161 in QR2 resulted in the total loss of the enzymatic activity towards activation of CB1954, whereas the rates of reduction towards menadione are not altered. Homo sapiens 16129418 1.10.99.2 N161H 1148 1332 Mutation of Asn161 to His161 in QR2 resulted in the total loss of the enzymatic activity towards activation of CB1954, whereas the rates of reduction towards menadione are not altered. Homo sapiens 16129418 1.10.99.2 N161H 1148 1332 Mutation of Asn161 to His161 in QR2 resulted in the total loss of the enzymatic activity towards activation of CB1954, whereas the rates of reduction towards menadione are not altered. Homo sapiens 16129418 1.10.99.2 N161H 1148 1332 Mutation of Asn161 to His161 in QR2 resulted in the total loss of the enzymatic activity towards activation of CB1954, whereas the rates of reduction towards menadione are not altered. Homo sapiens 16129418 1.10.99.2 N161H 20476 20644 The mutation of N161 to H161 would not affect the binding and orientation of menadione to QR2, as there is no possible clash between His161 with any atoms in menadione. Homo sapiens 16129418 1.10.99.2 N161H 21804 21899 The replacement of N161 by H161 will ˚ shorten the active site cavity by about 1.9 A (Fig. 4A). Homo sapiens 16129418 1.10.99.2 N161H 22992 23250 Since there is no substitution group at the 3 position in menadione, the mutation of Asn161 to His161 would not hinder the binding and positioning of menadione at the active site (Fig. 4B), and hence both QR1 and QR2 are active in the reduction of menadione. Homo sapiens 16129418 1.10.99.2 N161H 22992 23250 Since there is no substitution group at the 3 position in menadione, the mutation of Asn161 to His161 would not hinder the binding and positioning of menadione at the active site (Fig. 4B), and hence both QR1 and QR2 are active in the reduction of menadione. Homo sapiens 16129418 1.10.99.2 N161H 23251 23576 Mutation of Asn161 to histidine abolished the enzymatic activity of QR2 in the activation of CB1954 In order to confirm the importance of residue 161 in the differentiation of substrate specificities between QR1 and QR2, we generated a QR2 mutant in which residue Asn161 was mutated to histidine by site-directed mutagenesis. Homo sapiens 16129418 1.10.99.2 N161H 23251 23576 Mutation of Asn161 to histidine abolished the enzymatic activity of QR2 in the activation of CB1954 In order to confirm the importance of residue 161 in the differentiation of substrate specificities between QR1 and QR2, we generated a QR2 mutant in which residue Asn161 was mutated to histidine by site-directed mutagenesis. Homo sapiens 16129418 1.10.99.2 N161H 23577 23774 The mutant protein was expressed and purified similarly to the wild-type QR2, with no reduction of expression levels or alteration in the behavior on ion-exchange and size-exclusion chromatography. Homo sapiens 16129418 1.10.99.2 N161H 23577 23774 The mutant protein was expressed and purified similarly to the wild-type QR2, with no reduction of expression levels or alteration in the behavior on ion-exchange and size-exclusion chromatography. Homo sapiens 16129418 1.10.99.2 N161H 23775 23999 We tested the activity of the mutant protein in the activation of CB1954 using SUB10R as co-substrate, which was proved to be effective in the activation of CB1954 both in vitro and in vivo in a cellular toxicity assay [11]. Homo sapiens 16129418 1.10.99.2 N161H 23775 23999 We tested the activity of the mutant protein in the activation of CB1954 using SUB10R as co-substrate, which was proved to be effective in the activation of CB1954 both in vitro and in vivo in a cellular toxicity assay [11]. Homo sapiens 16129418 1.10.99.2 N161H 24000 24169 In comparison with the wild-type QR2, the mutant QR2 (N161H) exhibited no detectable activity in the reduction of CB1954 up to concentrations of 150 lM CB1954 (Fig. 5A). Homo sapiens 16129418 1.10.99.2 N161H 24000 24169 In comparison with the wild-type QR2, the mutant QR2 (N161H) exhibited no detectable activity in the reduction of CB1954 up to concentrations of 150 lM CB1954 (Fig. 5A). Homo sapiens 16129418 1.10.99.2 N161H 24486 24579 We further analyzed the catalytic activities of the mutant QR2 in the reduction of menadione. Homo sapiens 16129418 1.10.99.2 N161H 27652 27771 Comparison of mutant QR2 (Asn161 to His) with wild-type QR2 in the reduction of menadione and the activation of CB1954. Homo sapiens 16129418 1.10.99.2 N161H 27652 27771 Comparison of mutant QR2 (Asn161 to His) with wild-type QR2 in the reduction of menadione and the activation of CB1954. Homo sapiens 16129418 1.10.99.2 N161H 27773 27835 A) The mutant protein is inactive in the activation of CB1954. Homo sapiens 16129418 1.10.99.2 N161H 27773 27835 A) The mutant protein is inactive in the activation of CB1954. Homo sapiens 16129418 1.10.99.2 N161H 28359 28499 B) Reduction of menadione by wild-type QR2 (Ç) and mutant QR2 (h) was at the same rate when substrate SUB10R was used as the electron donor. Homo sapiens 16129418 1.10.99.2 N161H 28359 28499 B) Reduction of menadione by wild-type QR2 (Ç) and mutant QR2 (h) was at the same rate when substrate SUB10R was used as the electron donor. Homo sapiens 16129418 2.1.1.148 I65M/L175M 4670 4957 The enzymatic activity of both the wild-type and the I65M/L175M double mutant was confirmed by testing whether the MtbThyX protein could complement the defect in thymidylate synthesis of the E. coli strain, in TSdeficient c2913 E. coli in which the thyA gene has been deleted (Figure 1). Mycobacterium tuberculosis 16139296 2.1.1.148 I65M 18135 18362 Mutational studies on MtbThyX Three categories of directed mutations in the wild-type M. tuberculosis thyX gene were made and tested for the ability of the resultant variants to complement the TS-defective E. coli strain c2913. Mycobacterium tuberculosis 16139296 2.1.1.148 L175M 18135 18362 Mutational studies on MtbThyX Three categories of directed mutations in the wild-type M. tuberculosis thyX gene were made and tested for the ability of the resultant variants to complement the TS-defective E. coli strain c2913. Mycobacterium tuberculosis 16139296 2.1.1.148 L175M 18363 18628 First, the mutations I65M and L175M (both the single and double mutants) were created to have better phasing power in structure determination; these did not impair the enzyme activity and both encoded proteins supported the growth of E. coli c2913 strain (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 I65M 18363 18628 First, the mutations I65M and L175M (both the single and double mutants) were created to have better phasing power in structure determination; these did not impair the enzyme activity and both encoded proteins supported the growth of E. coli c2913 strain (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 S105E 18886 19004 As expected, the enzymes that carried H69E and S105E mutations failed to complement the E. coli c2913 cells (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 H69E 18886 19004 As expected, the enzymes that carried H69E and S105E mutations failed to complement the E. coli c2913 cells (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 H69E 19005 19228 These proteins can be expressed and isolated from E. coli, suggesting that the lack of complementation is a result of the mutation, and not of poor expression or instability of the mutant ThyX (J.E.U., unpublished results). Mycobacterium tuberculosis 16139296 2.1.1.148 S105E 19005 19228 These proteins can be expressed and isolated from E. coli, suggesting that the lack of complementation is a result of the mutation, and not of poor expression or instability of the mutant ThyX (J.E.U., unpublished results). Mycobacterium tuberculosis 16139296 2.1.1.148 R95A 19908 20047 Of these, the R95A and R95D mutants failed to complement the E. coli c2913 cells, whereas the R95K mutant supported their growth (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 R95D 19908 20047 Of these, the R95A and R95D mutants failed to complement the E. coli c2913 cells, whereas the R95K mutant supported their growth (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 Y108F 20448 20547 Replacement of Tyr108 by Phe yielded a mutant that did complement the growth of E. coli c2913 cells Mycobacterium tuberculosis 16139296 2.1.1.148 K165A 21100 21244 Both the K165A and R168A mutants of MtbThyX failed to complement growth of the E. coli c2913 strain lacking its own conventional ThyA (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 R168A 21100 21244 Both the K165A and R168A mutants of MtbThyX failed to complement growth of the E. coli c2913 strain lacking its own conventional ThyA (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 K165A 23760 23962 Our mutagenesis results indicate that Lys165 and Arg168 play a role in MtbThyX action, since changing these residues into Ala results in variants that cannot complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 R168A 23760 23962 Our mutagenesis results indicate that Lys165 and Arg168 play a role in MtbThyX action, since changing these residues into Ala results in variants that cannot complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 R95D 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 Y108F 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 K165A 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 L175M 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 H69E 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 R95A 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 R168A 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 R95K 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 I65M 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 S105E 29392 29583 Mutations created in MtbThyX Original and mutant amino acid in M. tuberculosis ThyX I65M L175M H69E S105E R95A R95D R95K Y108F K165A R168A Complementation in ThyAK E. coli C C K K K K C C K K Mycobacterium tuberculosis 16139296 2.1.1.148 R168A 37902 38027 Substitution of Lys165 and Arg168 by alanine abrogates the ability of MtbThyX to complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 K165A 37902 38027 Substitution of Lys165 and Arg168 by alanine abrogates the ability of MtbThyX to complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 K165A 37902 38027 Substitution of Lys165 and Arg168 by alanine abrogates the ability of MtbThyX to complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 R168A 37902 38027 Substitution of Lys165 and Arg168 by alanine abrogates the ability of MtbThyX to complement ThyA-defective E. coli (Table 4). Mycobacterium tuberculosis 16139296 2.1.1.148 I65M/L175M 40803 41174 Transformants were selected on LB agar plates with 30 mg/ml of kanamycin at 37 8C. Colonies from each of the ThyX mutants were streaked onto LB agar plates with kanamycin and grown overnight at 37 8C. Each of the different ThyX mutated strains was again streaked onto two different M9 minimal medium plates, one without thymidine and the other with 50 mg/ml of thymidine. Mycobacterium tuberculosis 16139296 2.1.1.148 I65M/L175M 40803 41174 Transformants were selected on LB agar plates with 30 mg/ml of kanamycin at 37 8C. Colonies from each of the ThyX mutants were streaked onto LB agar plates with kanamycin and grown overnight at 37 8C. Each of the different ThyX mutated strains was again streaked onto two different M9 minimal medium plates, one without thymidine and the other with 50 mg/ml of thymidine. Mycobacterium tuberculosis 16139296 2.1.1.148 I65M/L175M 42776 42955 Crystallization and data collection Crystals of the Ile65Met/Leu175Met mutant of MtbThyX were obtained by the sitting-drop, vapordiffusion method in the presence of 3-6 mM BrdUMP. Mycobacterium tuberculosis 16139296 1.9.3.1 E286Q 2265 2396 Several enzymes from different organisms (bovine, two bacterial enzymes, and several mutants) are compared and discussed in detail. Rhodobacter sphaeroides 16242114 1.9.3.1 E286Q 7908 8039 Several enzymes from different organisms (bovine, two bacterial enzymes, and several mutants) are compared and discussed in detail. Rhodobacter sphaeroides 16242114 1.9.3.1 E286Q 15508 15747 In the experiments, where Glu242 is mutated, or the D channel is blocked, the residual reduction of oxygen to water by CcO is observed [45] and this is attributed to the supply of protons from the P side of the membrane called ‘‘leaking’’. Rhodobacter sphaeroides 16242114 1.9.3.1 E286Q 24913 25075 Below, we discuss the experimental data reported recently for different systems and for several mutants, and compare these data with the predictions of the model. Rhodobacter sphaeroides 16242114 1.9.3.1 D132N 31677 31836 The small difference with the experiment might be due to several factors, including unaccounted conformational changes in the mutant enzyme with respect to WT. Rhodobacter sphaeroides 16242114 1.9.3.1 D124N 31677 31836 The small difference with the experiment might be due to several factors, including unaccounted conformational changes in the mutant enzyme with respect to WT. Rhodobacter sphaeroides 16242114 1.9.3.1 D124N 31956 32131 The conformational changes may be also responsible for the inhibition of both protonic phases of the F to O transition in the equivalent D124N mutant of P. denitrificans [21]. Rhodobacter sphaeroides 16242114 1.9.3.1 D124N 32132 32365 However, the more likely factor that could affect the difference between the calculated and observed amplitudes is the incomplete conversion of the mutant enzyme to the ferryl-oxo state F by the H2O2 treatment [17] in the experiment. Rhodobacter sphaeroides 16242114 1.9.3.1 D124N 32366 32510 Qualitatively, this effect results in the lower amplitudes in the mutant enzyme compared to WT, where the conversion is expected to be complete. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 32511 32753 Therefore, this effect should increase the ratio of the amplitudes of the fast phases of WT and the mutant, and could in principle be responsible for the difference between the theoretical factor 1.7 and experimentally observed factors 2 - 3. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 32511 32753 Therefore, this effect should increase the ratio of the amplitudes of the fast phases of WT and the mutant, and could in principle be responsible for the difference between the theoretical factor 1.7 and experimentally observed factors 2 - 3. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 32761 32945 N139D The N139D mutant does not pump protons, while the turnover rate, i.e., the rate of delivery of chemical protons to the catalytic center, is increased by a factor of 2- 3 [17,46]. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 32946 33138 In the N139D mutant the rates of proton transfer along the D channel, and in particular between E286 (E242 in bovine, our group g) and PLS (His291 in bovine, group b), are presumably modified. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 33311 33681 In terms of our model, in the mutant, the rate of proton transfer from E286 to PLS (group a) k 12 is presumably decreased, and/ or the rate of proton delivery to the binuclear center k 14 is increased (this assumption is supported by the observed increased turnover rate of the enzyme), so that the key assumption of the kinetic model, k 12>>k 14 is no longer satisfied. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 33311 33681 In terms of our model, in the mutant, the rate of proton transfer from E286 to PLS (group a) k 12 is presumably decreased, and/ or the rate of proton delivery to the binuclear center k 14 is increased (this assumption is supported by the observed increased turnover rate of the enzyme), so that the key assumption of the kinetic model, k 12>>k 14 is no longer satisfied. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 33311 33681 In terms of our model, in the mutant, the rate of proton transfer from E286 to PLS (group a) k 12 is presumably decreased, and/ or the rate of proton delivery to the binuclear center k 14 is increased (this assumption is supported by the observed increased turnover rate of the enzyme), so that the key assumption of the kinetic model, k 12>>k 14 is no longer satisfied. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 33311 33681 In terms of our model, in the mutant, the rate of proton transfer from E286 to PLS (group a) k 12 is presumably decreased, and/ or the rate of proton delivery to the binuclear center k 14 is increased (this assumption is supported by the observed increased turnover rate of the enzyme), so that the key assumption of the kinetic model, k 12>>k 14 is no longer satisfied. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 34499 34790 The data reported in Table 2 of their paper show that the amplitude of the single protonic phase in the mutant, corrected for the incompleteness of the reaction with hydrogen peroxide, is decreased by a factor of 1.3 with respect to the amplitude of the slow protonic phase in the WT enzyme. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 34499 34790 The data reported in Table 2 of their paper show that the amplitude of the single protonic phase in the mutant, corrected for the incompleteness of the reaction with hydrogen peroxide, is decreased by a factor of 1.3 with respect to the amplitude of the slow protonic phase in the WT enzyme. Rhodobacter sphaeroides 16242114 1.9.3.1 D132N 42182 42376 A reasonable agreement with five different experiments with both bovine and two bacterial enzymes, and three different mutants, provides a support for the proposed kinetic model of the CcO pump. Rhodobacter sphaeroides 16242114 1.9.3.1 D132N 42182 42376 A reasonable agreement with five different experiments with both bovine and two bacterial enzymes, and three different mutants, provides a support for the proposed kinetic model of the CcO pump. Rhodobacter sphaeroides 16242114 1.9.3.1 N139D 46256 46579 V f /V s V f /V s V f /V s V f /V s V f /V s V f /V s V f (WT)/ V f (D132N) V s(WT)/ ˜ V s(N139D) prot V slow /V elec prot V slow /V elec 0.45 0.62 0.60 0.53 0.51 0.45 1.70 1.6 4.6 3.2 ( =4 0.58 0.77 0.75 0.67 0.66 0.58 1.86 1.31 3.9 3.3 ( = 20 0.63 0.82 0.80 0.72 0.70 0.63 1.90 1.25 3.7 3.3 exp 0.4 0.5 0.4 0.33 2-3 1.3 ‘ Rhodobacter sphaeroides 16242114 5.3.3.1 E126L 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 N100S 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 N323L 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 H232A 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 N100A 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 S322A 10018 10283 Chou-Fasman and Garnier-Osguthorpe-Robson analysis of each mutant enzyme was used to choose amino acid substitutions that produced no apparent changes in the secondary structure of the protein (Protylze program, Scientific and Educational Software, State Line, PA). Homo sapiens 16889958 5.3.3.1 N323E 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 V87L 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 V87L 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 L236V 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 L236V 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 N323E 20929 21180 When the seven amino acids in the substrate binding shells in the 3␤HSD 1 model and the observed structure of 17␤-HSD 1 are compared, three are the same (Tyr154, His232, and Phe236) and three are similar in nature (Val87Leu, Leu236Val, and Asn323Glu). Homo sapiens 16889958 5.3.3.1 E126L 21181 21234 Only the Glu126Leu variation is a significant change. Homo sapiens 16889958 5.3.3.1 Y154F 24005 24131 Each of the single mutations Ser124Ala, Tyr154Phe, Lys158Gln results in the complete loss of dehydrogenase activity [8,36,37]. Homo sapiens 16889958 5.3.3.1 S124A 24005 24131 Each of the single mutations Ser124Ala, Tyr154Phe, Lys158Gln results in the complete loss of dehydrogenase activity [8,36,37]. Homo sapiens 16889958 5.3.3.1 K158Q 24005 24131 Each of the single mutations Ser124Ala, Tyr154Phe, Lys158Gln results in the complete loss of dehydrogenase activity [8,36,37]. Homo sapiens 16889958 5.3.3.1 D257L 24799 24910 Dehydrogenase and isomerase activities are completely abolished in the Asp257Leu and Asp258Leu mutants [30,36]. Homo sapiens 16889958 5.3.3.1 D258L 24799 24910 Dehydrogenase and isomerase activities are completely abolished in the Asp257Leu and Asp258Leu mutants [30,36]. Homo sapiens 16889958 5.3.3.1 H156Y 24911 25076 In our model, these Asp residues are exposed to possible interaction with water and their replacement with hydrophobic Leu residues is thermodynamically unfavorable. Homo sapiens 16889958 5.3.3.1 Q105M 24911 25076 In our model, these Asp residues are exposed to possible interaction with water and their replacement with hydrophobic Leu residues is thermodynamically unfavorable. Homo sapiens 16889958 5.3.3.1 H156Y 25173 25418 Destabilization of interactions at the dimer interface (Fig. 6) due to Gln105Met and His156Tyr mutations [8,35] was demonstrated in a dramatic increase in the substrate Km and inhibitor Ki values of 3␤-HSD 1 to equal those measured for 3␤-HSD 2. Homo sapiens 16889958 5.3.3.1 Q105M 25173 25418 Destabilization of interactions at the dimer interface (Fig. 6) due to Gln105Met and His156Tyr mutations [8,35] was demonstrated in a dramatic increase in the substrate Km and inhibitor Ki values of 3␤-HSD 1 to equal those measured for 3␤-HSD 2. Homo sapiens 16889958 5.3.3.1 H156Y 25173 25418 Destabilization of interactions at the dimer interface (Fig. 6) due to Gln105Met and His156Tyr mutations [8,35] was demonstrated in a dramatic increase in the substrate Km and inhibitor Ki values of 3␤-HSD 1 to equal those measured for 3␤-HSD 2. Homo sapiens 16889958 5.3.3.1 Q105M 25173 25418 Destabilization of interactions at the dimer interface (Fig. 6) due to Gln105Met and His156Tyr mutations [8,35] was demonstrated in a dramatic increase in the substrate Km and inhibitor Ki values of 3␤-HSD 1 to equal those measured for 3␤-HSD 2. Homo sapiens 16889958 5.3.3.1 H156Y 25636 25777 This Hbond is disrupted in 3␤-HSD 1 by the replacement of Gln105 with Met and His156 with Tyr, which is the residue present in 3␤-HSD 2 [35]. Homo sapiens 16889958 5.3.3.1 Q105M 25636 25777 This Hbond is disrupted in 3␤-HSD 1 by the replacement of Gln105 with Met and His156 with Tyr, which is the residue present in 3␤-HSD 2 [35]. Homo sapiens 16889958 5.3.3.1 D35A/K36R 26054 26224 The double mutant has no 3␤-HSD or isomerase activity in the presence of NAD(H) and both activities are found with three-fold decrease in Vmax in the presence of NADP(H). Homo sapiens 16889958 5.3.3.1 N323L 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 S322A 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 N100A 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 H232A 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 E126L 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 N100S 26702 26836 This deletion mutant of 3␤-HSD 1 had substrate and cofactor kinetics that were almost identical to those of the wild-type enzyme [39]. Homo sapiens 16889958 5.3.3.1 S322A 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 N323L 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 H232A 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 N100A 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 E126L 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 N100S 26838 27039 Rationally designed mutants based upon the model that successfully elucidate the mechanism of the dual activity of 3␤-HSD 1 and permit the design of selective inhibitors are the best test of the model. Homo sapiens 16889958 5.3.3.1 N100S 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 N100A 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 N100S 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 N100A 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 N100S 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 N100A 28160 28299 Replacement of Asn100 disrupts the network of H-bonds resulting in the loss of oxidoreductase activity and reduction of isomerase activity. Homo sapiens 16889958 5.3.3.1 H232A 31747 31913 The His232Ala mutation causes the complete loss of dehydrogenase activity and a significant reduction in isomerase activity based on the kcat values (Tables 2 and 3). Homo sapiens 16889958 5.3.3.1 H232A 31747 31913 The His232Ala mutation causes the complete loss of dehydrogenase activity and a significant reduction in isomerase activity based on the kcat values (Tables 2 and 3). Homo sapiens 16889958 5.3.3.1 N323L 31914 32090 For the Asn323Leu mutant the Km values for both 3␤-HSD and isomerase substrate utilization increased greatly indicating a corresponding reduction of binding affinity (Table 2). Homo sapiens 16889958 5.3.3.1 N323L 31914 32090 For the Asn323Leu mutant the Km values for both 3␤-HSD and isomerase substrate utilization increased greatly indicating a corresponding reduction of binding affinity (Table 2). Homo sapiens 16889958 5.3.3.1 H232A 32091 32233 However, the Km values of His232Ala and Asn323Leu for the NADH cofactor are similar to the values measured for the wild-type enzyme (Table 3). Homo sapiens 16889958 5.3.3.1 N323L 32091 32233 However, the Km values of His232Ala and Asn323Leu for the NADH cofactor are similar to the values measured for the wild-type enzyme (Table 3). Homo sapiens 16889958 5.3.3.1 S322A 32443 32648 The reduction of both activities for the Ser322Ala mutant may indicate its direct involvement in substrate recognition or an indirect effect on the critically important neighboring Asn323 (Tables 2 and 3). Homo sapiens 16889958 5.3.3.1 E126L 32649 32820 Kinetic studies of the Glu126Leu mutant show a dramatic reduction of the isomerase activity, suggesting direct participation of Glu126 in the isomerase reaction (Table 2). Homo sapiens 16889958 5.3.3.1 E126L 32978 33120 The retention of a trace of isomerase activity in the mutant may suggest an additional, less effective mechanism of proton transfer via water. Homo sapiens 16889958 5.3.3.1 Y154F 33518 33664 However, a four-fold reduction of the corresponding kcat value for the isomerase reaction in the Tyr154Phe mutant with significant residual activ- Homo sapiens 16889958 5.3.3.1 Y154F 33518 33664 However, a four-fold reduction of the corresponding kcat value for the isomerase reaction in the Tyr154Phe mutant with significant residual activ- Homo sapiens 16889958 1.1.1.9 E154C 1162 1387 E154C retained 0.019 + 0.003 % and 0.74 + 0.03 % of − − wild-type catalytic efficiency (kcat /K sorbitol = 7800 + 700 M−1 · s−1 ) − −1 + and kcat (=161 + 4 s ) for NAD -dependent oxidation of sorbitol − at 25 ◦C respectively. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 1388 1640 The pH profile of kcat /K sorbitol for E154C decreased below an apparent pK of 9.1 + 0.3, reflecting a shift in − pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 1388 1640 The pH profile of kcat /K sorbitol for E154C decreased below an apparent pK of 9.1 + 0.3, reflecting a shift in − pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 1891 2159 Under conditions eliminating their different pHdependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7 + 0.2 − (E154C, 1.7 + 0.1) and 1.9 + 0.3 (E154C, 2.4 + 0.2) on kcat /K sorbitol − − − + respectively. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 1891 2159 Under conditions eliminating their different pHdependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7 + 0.2 − (E154C, 1.7 + 0.1) and 1.9 + 0.3 (E154C, 2.4 + 0.2) on kcat /K sorbitol − − − + respectively. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 8031 8431 Kinetic evidence for a Glu → Cys variant of rat SDH, reported by Karlson and H¨ og [8], is also consistent with o¨ a major function of glutamic acid in substrate binding because the site-directed replacement caused a large (500-fold) decrease in sorbitol binding affinity (K sorbitol ), whereas the catalytic centre activity (kcat ) of the mutant was even ≈ 4 times higher than that of the wild-type. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 8031 8431 Kinetic evidence for a Glu → Cys variant of rat SDH, reported by Karlson and H¨ og [8], is also consistent with o¨ a major function of glutamic acid in substrate binding because the site-directed replacement caused a large (500-fold) decrease in sorbitol binding affinity (K sorbitol ), whereas the catalytic centre activity (kcat ) of the mutant was even ≈ 4 times higher than that of the wild-type. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 8857 9214 A second possibility is that the replacement of the thiol group ligand in ADH by a carboxy [7] or a H2 O/hydroxy group ligand [5] in PDH alters the electrophilic character of Zn2+ and therefore, changes the propensity of the metal ion to bind alcohol and stabilize the proposed alkoxide intermediate state of the reaction through inner-sphere co-ordination. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 8857 9214 A second possibility is that the replacement of the thiol group ligand in ADH by a carboxy [7] or a H2 O/hydroxy group ligand [5] in PDH alters the electrophilic character of Zn2+ and therefore, changes the propensity of the metal ion to bind alcohol and stabilize the proposed alkoxide intermediate state of the reaction through inner-sphere co-ordination. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 11847 11989 E154C was isolated using a modified twostep procedure consisting of dye-ligand affinity chromatography followed by preparative gel filtration. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 11990 12163 To avoid contamination of E154C by wild-type enzyme, all purifications of the mutant were carried out with Procion Red HE3B (Red 120) freshly immobilized on Sepharose 4B-CL. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 13116 13346 Apparent kinetic parameters were determined at a constant saturating concentrations of sorbitol (300 mM for GmXDH, 25 mM for slSDH and 2 M for E154C) or NAD+ (10 mM for all enzymes), and various concentrations of NAD+ or sorbitol. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 15781 16013 Variation in ionic strength across the chosen pL range was small (I ≈ 0.10 M), and control experiments in which 25 mM NaCl was added to the assays revealed the absence of significant ionic strength effects under the conditions used. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 20032 20395 Eqn (3) describes linear competitive inhibition where [I] is the inhibitor concentration and K i is the apparent dissociation constant of I. [E] was calculated from a molecular mass of the enzyme subunit of 37.8 kDa and was based on measurements of protein concentration (slSDH) or the concentration of Zn2+ associated with the protein preparation (GmXDH; E154C). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 21237 21322 the same, within limits of experimental error, in wild-type and E154C forms of GmXDH. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 21474 21610 Purification of E154C from the E. coli cell extract required a protocol different from that utilized for isolation of the wildtype [18]. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 22047 22329 Supplementary Figure S4 (at http://www.BiochemJ.org/bj/404/bj4040421add.htm) shows SDS/PAGE analysis of purified E154C. The apparent molecular mass of the subunit of E154C was 38.7 kDa, which is in agreement with a molecular mass of 37.4 kDa calculated from the amino acid sequence. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 22330 22523 Purified E154C eluted from an analytical gel-filtration column as a single protein peak with an apparent molecular mass of 160 kDa that contained all of the applied protein and enzyme activity. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 22524 22612 In a similar manner to the wild-type, the mutant seemed to be a functional homotetramer. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 22613 22831 Different preparations of E154C (n = 4) that appeared homogeneous in SDS/PAGE (see Supplementary Figure S4 at http://www.BiochemJ.org/bj/404/ bj4040421add.htm) contained between 0.1 and 0.4 atom Zn2+ / protein subunit. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 22832 23041 Although the source of this variable Zn2+ content remained unknown, the activity of E154C solutions based on the molarity of Zn2+ -containing active sites was constant across the different enzyme preparations. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 23042 23242 Exogenous Zn2+ (added as ZnSO4 ) in the concentration range 1-100 µM was a strong irreversible inhibitor of wild-type and the mutant, the IC50 value of about 5 + 1 µM being identical for both enzymes. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 23243 23446 Note that − E154C was stable during the initial rate assays described below, suggesting that inactivation due to release of the active-site Zn2+ did not occur in the time-span of the kinetic experiments. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 23243 23446 Note that − E154C was stable during the initial rate assays described below, suggesting that inactivation due to release of the active-site Zn2+ did not occur in the time-span of the kinetic experiments. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 24302 24520 The pL profiles of kcat /K sorbitol for E154C in 1 H2 O and 2 H2 O also displayed a decrease in rate at low pL but, unlike wild-type GmXDH and slSDH, the activity of the mutant was lost completely below pK (Figure 2D). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 24302 24520 The pL profiles of kcat /K sorbitol for E154C in 1 H2 O and 2 H2 O also displayed a decrease in rate at low pL but, unlike wild-type GmXDH and slSDH, the activity of the mutant was lost completely below pK (Figure 2D). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 24302 24520 The pL profiles of kcat /K sorbitol for E154C in 1 H2 O and 2 H2 O also displayed a decrease in rate at low pL but, unlike wild-type GmXDH and slSDH, the activity of the mutant was lost completely below pK (Figure 2D). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 25657 25963 Solvent KIEs were measured at pL 9.0 (slSDH, wild-type GmXDH) or pL 10.0 (E154C) where pL profiles of kcat and kcat /K sorbitol approach plateau values and thus differences in pL profiles of the respective kinetic parameter in 1 H2 O and 2 H2 O are eliminated (wild-type GmXDH; slSDH) or minimized (E154C). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 25657 25963 Solvent KIEs were measured at pL 9.0 (slSDH, wild-type GmXDH) or pL 10.0 (E154C) where pL profiles of kcat and kcat /K sorbitol approach plateau values and thus differences in pL profiles of the respective kinetic parameter in 1 H2 O and 2 H2 O are eliminated (wild-type GmXDH; slSDH) or minimized (E154C). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 31801 31922 Purified E154C was shown to have retained a functional Zn2+ centre that displayed fractional saturation with Zn2+ of 0.4. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 31923 32186 The catalytic efficiency of the mutant for NAD+ dependent oxidation of sorbitol at pH 9.0 was decreased 103.7 -fold in comparison with the corresponding wild-type value, suggesting that Glu154 is relatively more important for catalytic function than Zn2+ binding. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 32773 32964 The Zn2+ centre of human liver SDH differs from that of horse liver ADH mainly in the substitution of the Cys174 ligand [4] by a water molecule linked through the secondary sphere Glu155 [5]. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 37672 37842 The pH-dependence of kcat /K sorbitol for E154C was clearly distinguished from that for the wild-type, showing an upshift in pK by + 1.7 pH units to a value of 9.1 + 0.3. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 37951 38111 The pH profile of the mutant did not decrease to an asymptote level at low pH, suggesting that unlike the wildtype, the protonated form of E154C does not react. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 37951 38111 The pH profile of the mutant did not decrease to an asymptote level at low pH, suggesting that unlike the wildtype, the protonated form of E154C does not react. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 38112 38336 Considering the pK of + 0.2 in 2 H2 O solvent, the observable pK values of 7.4 + 0.1 and 9.1 + 0.3 in the NAD+ complexes of wild-type and − − E154C forms of GmXDH respectively are tentatively assigned to a Zn2+ -bound water. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 38337 38634 Mutation of Glu154 did not abolish the pH-dependence because Zn2+ -water is still expected to be present in E154C. However, the carboxylate side chain of the glutamic acid residue could facilitate deprotonation of Zn2+ -water through a proton relay or hydrogen bonding, whereas Cys154 , due to the Galactocandida mastotermitis 17343568 1.1.1.9 E154C 39709 39855 However, the differences in D kcat /K sorbitol for E154C and wild-type at pH 9.0 appear to reflect solely the altered pH-dependence in the mutant. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 39709 39855 However, the differences in D kcat /K sorbitol for E154C and wild-type at pH 9.0 appear to reflect solely the altered pH-dependence in the mutant. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 39857 40013 D kcat /K sorbitol ) values are similar at the pK of the wild-type enzymecatalysed reaction (pH 7.5) and at the pK of the E154C-catalysed reaction (pH 9.0). Galactocandida mastotermitis 17343568 1.1.1.9 E154C 40719 40959 Under conditions where kcat /K sorbitol did not show a pL-dependence, values of D2O kcat /K sorbitol for wild-type and E154C forms of GmXDH were almost identical, suggesting that Glu154 is not directly involved in catalytic proton transfer. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 40960 41157 If it were, a Glu → Cys substitution would arguably make the proton transfer more rate-limiting, detectable as a significant increase in D2O kcat /K sorbitol for E154C in comparison with wild-type. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 40960 41157 If it were, a Glu → Cys substitution would arguably make the proton transfer more rate-limiting, detectable as a significant increase in D2O kcat /K sorbitol for E154C in comparison with wild-type. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 45239 45387 Below the pK of about 7.2-7.4 in enzyme-NAD+ , Zn2+ -water is protonated, causing a large decrease in activity in both wild-type and mutant enzymes. Galactocandida mastotermitis 17343568 1.1.1.9 E154C 45388 45487 However, loss of activity is only partial in wild-type, whereas it is complete in the E154C mutant. Galactocandida mastotermitis 17343568 1.1.1.102 A175T 1276 1403 The identified mutation is limited to SMA-affected animals and carriers and always appears in context of the founder haplotype. Bos taurus 17420465 1.1.1.102 A175T 1538 1618 Importantly, the Thr variant found in SMA animals showed no detectable activity. Bos taurus 17420465 1.1.1.102 A175T 1619 1769 Surprisingly, in an in vivo assay the mutated gene complements the growth defect of a homologous yeast knockout strain as well as the healthy variant. Bos taurus 17420465 1.1.1.102 A175T 5631 5788 Thus, bovine SMA and a similar human disease with identical name and neuropathological characteristics are caused by mutations in completely different genes. Bos taurus 17420465 1.1.1.102 A175T 5631 5788 Thus, bovine SMA and a similar human disease with identical name and neuropathological characteristics are caused by mutations in completely different genes. Bos taurus 17420465 1.1.1.102 A175T 5631 5788 Thus, bovine SMA and a similar human disease with identical name and neuropathological characteristics are caused by mutations in completely different genes. Bos taurus 17420465 1.1.1.102 A175T 8593 8843 Analyzing this complex pedigree with three-point analysis (the LINKMAP option of FASTLINK) we identified a significant linkage between SMA and the seven most distal markers on BTA24: BCL2࿝MS-LMU2410FVT1࿝A175T-LMU2404-LMU2403-LMU2407-DIK4971 (Fig. 1). Bos taurus 17420465 1.1.1.102 A175T 8844 8984 The most tightly linked marker interval is FVT1࿝A175T-LMU2404, with a logarithm of odds (LOD) score of 38.652 (Fig. 1, all affected calves). Bos taurus 17420465 1.1.1.102 A175T 11661 11907 The three-point analysis without two probable phenocopies mapped SMA with the highest LOD score (45.48) accurately to the SNP FVT1࿝A175T. The three-unit LOD score support interval covered the segments between markers LMU2410 and LMU2404 (Fig. 1). Bos taurus 17420465 1.1.1.102 A175T 13074 13216 The cDNAs were identical in length between the different animals and tissues, so we excluded the presence of splice-site mutations in introns. Bos taurus 17420465 1.1.1.102 A175T 13074 13216 The cDNAs were identical in length between the different animals and tissues, so we excluded the presence of splice-site mutations in introns. Bos taurus 17420465 1.1.1.102 A175T 14175 14337 We typed 122 affected animals with clinically and neuropathologically confirmed SMA diagnosis and found 119 of them were homozygous for the Thr variant (Thr-175). Bos taurus 17420465 1.1.1.102 A175T 17647 17875 In the majority of the cases a strong genetic bottleneck can eliminate some minor alleles in concerned subpopulation, but occasionally a bottleneck can amplify new mutations or minor alleles present in important founder animals. Bos taurus 17420465 1.1.1.102 A175T 17647 17875 In the majority of the cases a strong genetic bottleneck can eliminate some minor alleles in concerned subpopulation, but occasionally a bottleneck can amplify new mutations or minor alleles present in important founder animals. Bos taurus 17420465 1.1.1.102 A175T 18279 18585 To estimate the allele frequency of the Thr-175 variant in the modern German Braunvieh population, i.e., Braunvieh upgraded by American Brown Swiss, we genotyped 703 animals representing two different generations: 217 animals born between 1985 and 1990 and 486 animals born between 2000 and 2006 (Table 1). Bos taurus 17420465 1.1.1.102 A175T 18586 18762 During approximately three cattle generations (15 years) the allele frequency of the Thr-175 variant increased from 1.84% to 4.42% in the current genetically active population. Bos taurus 17420465 1.1.1.102 A175T 20784 21123 Because of the absence of clearly observed recombination in the interval rs29017741 to DIK4971 and reduced informativity of LMU2404 and LMU2403 (most common marker allele in phase with SMA allele) there still exists the possibility that the Thr-175 mutation is not causative but in perfect linkage disequilibrium with the causative allele. Bos taurus 17420465 1.1.1.102 A175T 21664 21823 Both the Thr-175 variant from an affected calf and the normal Ala-175 are expressed and purified in parallel under the same conditions with comparable results. Bos taurus 17420465 1.1.1.102 A175T 21664 21823 Both the Thr-175 variant from an affected calf and the normal Ala-175 are expressed and purified in parallel under the same conditions with comparable results. Bos taurus 17420465 1.1.1.102 A175T 24385 24636 In contrast, the same amount of the Thr-175 variant of FVT1, expressed and purified in parallel with the Ala-175 version, did not show any detectable enzymatic activity as analyzed by the appearance of the reaction product dihydrospingosine (Fig. 4C). Bos taurus 17420465 1.1.1.102 A175T 24385 24636 In contrast, the same amount of the Thr-175 variant of FVT1, expressed and purified in parallel with the Ala-175 version, did not show any detectable enzymatic activity as analyzed by the appearance of the reaction product dihydrospingosine (Fig. 4C). Bos taurus 17420465 1.1.1.102 A175T 24732 24966 The fact that the observed variation not only cosegregates with the SMA phenotype but also leads to a loss of enzymatic activity in vitro is a very strong indication that the described Ala-to-Thr exchange is indeed causing bovine SMA. Bos taurus 17420465 1.1.1.102 A175T 25180 25291 Therefore, we wanted to assess whether the observed loss-of-function mutation in FVT1 is also relevant in vivo. Bos taurus 17420465 1.1.1.102 A175T 25817 25944 tsc10 cells expressing the SMA variant fvt1-A175T show the same growth phenotype as those complemented by normal FVT1 (Fig. 5). Bos taurus 17420465 1.1.1.102 A175T 25945 26034 Thus, in vivo the A175T mutation in FVT1 does not lead to loss of KDS reductase activity. Bos taurus 17420465 1.1.1.102 A175T 26388 26486 Thus, the Thr-175 variant is not completely inactive but can carry out its basic function in vivo. Bos taurus 17420465 1.1.1.102 A175T 26832 27048 Mutation of the serine palmitoyl transferase, the enzyme that produces the substrate for FVT1, has been shown to cause hereditary sensory neuropathy (15), a neurodegenerative disorder that also affects motor neurons. Bos taurus 17420465 1.1.1.102 A175T 27287 27398 The FVT1 and the fvt1-A175T mutation show an intermediate complementation of the S. cerevisiae ⌬tsc10 knockout. Bos taurus 17420465 1.1.1.102 A175T 28393 28549 We speculate that the nearly inactive FVT1 variant observed as the cause of SMA in cattle leads to limited availability of ceramide and derived metabolites. Bos taurus 17420465 1.1.1.102 A175T 41683 41879 The plasmids pAK80-W0671 (FVT1) and pAK80-SMA031 ( fvt1-A175T) were obtained by amplification of the FVT1 cDNA from a control (W0671) and an affected animal (SMA031) and ligation into pGEM-T easy. Bos taurus 17420465 1.13.11.19 H95A 12669 12870 An H95A mutant of the wild-type pCMV-3 ϫ FLAG ADO construct was also generated by using the QuikChange II sitedirected mutagenesis kit and the same primer set used to mutate the pET SUMO ADO construct. Mus musculus 17581819 1.13.11.19 H95A 20453 20570 Wild-type and H95A ADO proteins were purified in an identical fashion and exhibited similar chromatographic profiles. Mus musculus 17581819 1.13.11.19 H95A 20453 20570 Wild-type and H95A ADO proteins were purified in an identical fashion and exhibited similar chromatographic profiles. Mus musculus 17581819 1.13.11.19 H95A 28610 28752 Because this mutated protein lacks an important putative metal binding residue, we predicted that the protein would be catalytically inactive. Mus musculus 17581819 1.13.11.19 H95A 28753 28956 Indeed, the H95A mutant did not show any detectable cysteamine dioxygenase activity when incubated with 8 mM cysteamine (limit of assay detection was ∼18 nmol·mg-1·min-1 or ∼0.8% of WT maximal activity). Mus musculus 17581819 1.13.11.19 H95A 28957 29093 The absence of activity was consistent with the metal analysis of the mutant protein, which showed it to be devoid of metal (see below). Mus musculus 17581819 1.13.11.19 H95A 31250 31550 Graphite molecule 2-mercaptoethanol, which differs from cysteamine furnace atomic absorption spectrometry also confirmed that by the substitution of the amine group with a hydroxyl the occupancy of iron in wild-type ADO protein was greater group, was not an effective inhibitor of activity (Fig. 4F). Mus musculus 17581819 1.13.11.19 H95A 31250 31550 Graphite molecule 2-mercaptoethanol, which differs from cysteamine furnace atomic absorption spectrometry also confirmed that by the substitution of the amine group with a hydroxyl the occupancy of iron in wild-type ADO protein was greater group, was not an effective inhibitor of activity (Fig. 4F). Mus musculus 17581819 1.13.11.19 H95A 31561 31887 To test the specificity of iron binding as well as its requireCollectively, these data suggest that WT ADO does not indiscriminately oxidize thiol-containing compounds but, ment for cysteamine dioxygenase activity, we conducted a instead, shows a high degree of specificity for the dioxygen- metal analysis on the H95A mutant. Mus musculus 17581819 1.13.11.19 H95A 32426 32495 This iron, the H95A mutant displayed no detectable catalytic activity Mus musculus 17581819 1.13.11.19 H95A 34601 34875 To accomplish this objective, we transiently transfected 3ϫ FLAG-tagged forms of catalytically active WT or catalytically incompetent H95A ADO into the human hepatoma HepG2/C3A cell line and then assayed for changes in the capacity of hypotaurine production from cysteamine. Mus musculus 17581819 1.13.11.19 H95A 35508 35646 Transfection with the catalytically inactive mutant, on the other hand, had no significant effect on intracellular hypotaurine production. Mus musculus 17581819 1.13.11.19 H95A 35508 35646 Transfection with the catalytically inactive mutant, on the other hand, had no significant effect on intracellular hypotaurine production. Mus musculus 17581819 1.13.11.19 H95A 39313 39464 Elimination of iron binding by mutation of His-95, a strictly conserved putative metal coordinating residue, produced a catalytically inactive protein. Mus musculus 17581819 2.2.1.1 H26A/H261A 46540 46908 As the donor substrates’ apparent KM values are virtually unchanged in the variant when compared to those of the wild-type enzyme, the low occupancy with covalent donor-ThDP adduct in equilibrium should not result from a diminished bimolecular binding of the substrate to form the Michaelis complex but rather from a higher kinetic barrier for covalent bond formation. Escherichia coli 17914867 2.2.1.1 H26A/H261A 46540 46908 As the donor substrates’ apparent KM values are virtually unchanged in the variant when compared to those of the wild-type enzyme, the low occupancy with covalent donor-ThDP adduct in equilibrium should not result from a diminished bimolecular binding of the substrate to form the Michaelis complex but rather from a higher kinetic barrier for covalent bond formation. Escherichia coli 17914867 2.2.1.1 H26A/H261A 46540 46908 As the donor substrates’ apparent KM values are virtually unchanged in the variant when compared to those of the wild-type enzyme, the low occupancy with covalent donor-ThDP adduct in equilibrium should not result from a diminished bimolecular binding of the substrate to form the Michaelis complex but rather from a higher kinetic barrier for covalent bond formation. Escherichia coli 17914867 2.2.1.1 H26A/H261A 46540 46908 As the donor substrates’ apparent KM values are virtually unchanged in the variant when compared to those of the wild-type enzyme, the low occupancy with covalent donor-ThDP adduct in equilibrium should not result from a diminished bimolecular binding of the substrate to form the Michaelis complex but rather from a higher kinetic barrier for covalent bond formation. Escherichia coli 17914867 2.2.1.1 H26A/H261A 46540 46908 As the donor substrates’ apparent KM values are virtually unchanged in the variant when compared to those of the wild-type enzyme, the low occupancy with covalent donor-ThDP adduct in equilibrium should not result from a diminished bimolecular binding of the substrate to form the Michaelis complex but rather from a higher kinetic barrier for covalent bond formation. Escherichia coli 17914867 1.1.1.14 Y110F 873 1357 Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 873 1357 Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 873 1357 Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 4803 4964 Equilibrium constants in vitro between three different quaternary states: monomer, dimer and tetramer in wild-type (wt) SDH and in Tyr110Phe SDH were determined. Rattus norvegicus 17952367 1.1.1.14 Y110F 6820 6942 Tyr110Phe SDH did not show a measurable activity at any stage of purification even at protein concentration of 0.1 mg/ ml. Rattus norvegicus 17952367 1.1.1.14 Y110F 8214 8339 All plasmids containing mutated fragments were checked by DNA sequence analyses for correct introduction of base alterations. Rattus norvegicus 17952367 1.1.1.14 Y110F 11124 11573 The change in Gibbs energy (DG’0) in the gel filtration experiments is calculated from the equilibrium constant (K’0) with Eq. 2, after 60 min of incubation at in vitro conditions of 10 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 0.1 mg/ml isolated protein and at temperatures 277-317 K. The difference in Gibbs energy between wt SDH and Tyr110Phe SDH at each temperature is given by: DDG’01 = DG’01wt À DG’01Tyr110Phe DDG’ 2 = DG’ 2 À DG’ 2 0 0 wt 0 Tyr110Phe Rattus norvegicus 17952367 1.1.1.14 Y110F 11590 11777 gives the change in Gibbs energy of the mutation between the monomeric and dimeric states and Eq. 4 gives the change in Gibbs energy of the mutation between dimeric and tetrameric states. Rattus norvegicus 17952367 1.1.1.14 Y110F 11590 11777 gives the change in Gibbs energy of the mutation between the monomeric and dimeric states and Eq. 4 gives the change in Gibbs energy of the mutation between dimeric and tetrameric states. Rattus norvegicus 17952367 1.1.1.14 Y110F 11590 11777 gives the change in Gibbs energy of the mutation between the monomeric and dimeric states and Eq. 4 gives the change in Gibbs energy of the mutation between dimeric and tetrameric states. Rattus norvegicus 17952367 1.1.1.14 Y110F 11590 11777 gives the change in Gibbs energy of the mutation between the monomeric and dimeric states and Eq. 4 gives the change in Gibbs energy of the mutation between dimeric and tetrameric states. Rattus norvegicus 17952367 1.1.1.14 Y110F 15010 15215 The relative protein concentrations for monomeric, dimeric and tetrameric wt SDH and Tyr110Phe SDH were determined with gel filtration experiments at five different temperatures from 277 to 317 K (Fig. 3). Rattus norvegicus 17952367 1.1.1.14 Y110F 15010 15215 The relative protein concentrations for monomeric, dimeric and tetrameric wt SDH and Tyr110Phe SDH were determined with gel filtration experiments at five different temperatures from 277 to 317 K (Fig. 3). Rattus norvegicus 17952367 1.1.1.14 Y110F 15010 15215 The relative protein concentrations for monomeric, dimeric and tetrameric wt SDH and Tyr110Phe SDH were determined with gel filtration experiments at five different temperatures from 277 to 317 K (Fig. 3). Rattus norvegicus 17952367 1.1.1.14 Y110F 15010 15215 The relative protein concentrations for monomeric, dimeric and tetrameric wt SDH and Tyr110Phe SDH were determined with gel filtration experiments at five different temperatures from 277 to 317 K (Fig. 3). Rattus norvegicus 17952367 1.1.1.14 Y110F 15379 15562 Tyr110Phe SDH was divided into tetramers, dimers and monomers at all temperatures investigated, with the relative amount of tetramers decreasing with increasing temperature (Fig. 3b). Rattus norvegicus 17952367 1.1.1.14 Y110F 15379 15562 Tyr110Phe SDH was divided into tetramers, dimers and monomers at all temperatures investigated, with the relative amount of tetramers decreasing with increasing temperature (Fig. 3b). Rattus norvegicus 17952367 1.1.1.14 Y110F 15563 15726 The changes in Gibbs energy (DDG’0) were calculated by comparison of the equilibrium constants between the different quaternary states in wt SDH and Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 15727 15904 The DDG’01 for the difference in the free energy of monomers and dimers between wt SDH and Tyr110Phe SDH was 1.4 kJ/mol at 307 K and 3.1 kJ/mol at 317 K (Table 1), respectively. Rattus norvegicus 17952367 1.1.1.14 Y110F 15905 16079 The DDG’02 for the difference in free energy of dimers and tetramers between wt SDH and Tyr110Phe SDH was 8.2 kJ/mol at 307 K and 8.1 kJ/mol at 317 K (Table 1), respectively. Rattus norvegicus 17952367 1.1.1.14 Y110F 16080 16220 No enzymatic activity was detectable, even at protein concentrations up to 0.1 mg/ml for Tyr110Phe SDH with the tested substrates (Table 2). Rattus norvegicus 17952367 1.1.1.14 Y110F 16221 16352 The circular dichroism analysis of wt SDH and Tyr110Phe SDH was similar and showed typical patterns of ordered secondary structure. Rattus norvegicus 17952367 1.1.1.14 Y110F 16926 17103 In the ionexchange chromatography step the two proteins showed identical elution profiles, but in the final gel filtration step Tyr110Phe SDH was divided into several fractions. Rattus norvegicus 17952367 1.1.1.14 Y110F 17104 17330 The homogenous tetrameric wt SDH and Tyr110Phe SDH fractions from the gel filtration purification step were collected and used for further catalytic activity measurements, circular dichroism analysis and stability experiments. Rattus norvegicus 17952367 1.1.1.14 Y110F 17331 17580 Calibration of the gel filtration column showed that the fractions from Tyr110Phe SDH corresponded to molecular sizes of >300-, 160-, 80- and 40-kDa proteins, which matches the sizes of aggregate, tetrameric, dimeric and monomeric SDH, respectively. Rattus norvegicus 17952367 1.1.1.14 Y110F 17331 17580 Calibration of the gel filtration column showed that the fractions from Tyr110Phe SDH corresponded to molecular sizes of >300-, 160-, 80- and 40-kDa proteins, which matches the sizes of aggregate, tetrameric, dimeric and monomeric SDH, respectively. Rattus norvegicus 17952367 1.1.1.14 Y110F 17331 17580 Calibration of the gel filtration column showed that the fractions from Tyr110Phe SDH corresponded to molecular sizes of >300-, 160-, 80- and 40-kDa proteins, which matches the sizes of aggregate, tetrameric, dimeric and monomeric SDH, respectively. Rattus norvegicus 17952367 1.1.1.14 Y110F 17837 17970 Changes in the relative concentration between the three states in wild-type (wt) SDH and Tyr110Phe SDH induced by temperature shifts. Rattus norvegicus 17952367 1.1.1.14 Y110F 18227 18320 Five different temperatures were measured from 277 K to 317 K. (a) wt SDH, (b) Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 19792 19986 The change in Gibbs energy in the comparison of the stability of different states (monomeric, dimeric and tetrameric states) by the mutation of wt SDH to Tyr110Phe SDH are given as DDG’0 values. Rattus norvegicus 17952367 1.1.1.14 Y110F 19792 19986 The change in Gibbs energy in the comparison of the stability of different states (monomeric, dimeric and tetrameric states) by the mutation of wt SDH to Tyr110Phe SDH are given as DDG’0 values. Rattus norvegicus 17952367 1.1.1.14 Y110F 19792 19986 The change in Gibbs energy in the comparison of the stability of different states (monomeric, dimeric and tetrameric states) by the mutation of wt SDH to Tyr110Phe SDH are given as DDG’0 values. Rattus norvegicus 17952367 1.1.1.14 Y110F 19792 19986 The change in Gibbs energy in the comparison of the stability of different states (monomeric, dimeric and tetrameric states) by the mutation of wt SDH to Tyr110Phe SDH are given as DDG’0 values. Rattus norvegicus 17952367 1.1.1.14 Y110F 23899 24129 The in vitro determined equilibrium constant between the monomeric and dimeric state is only slightly changed by the mutation of Tyr110 into Phe in SDH, with a corresponding change in Gibbs energy (DDG’01) of 1-3 kJ/mol (Table 1). Rattus norvegicus 17952367 1.1.1.14 Y110F 23899 24129 The in vitro determined equilibrium constant between the monomeric and dimeric state is only slightly changed by the mutation of Tyr110 into Phe in SDH, with a corresponding change in Gibbs energy (DDG’01) of 1-3 kJ/mol (Table 1). Rattus norvegicus 17952367 1.1.1.14 Y110F 24130 24295 However, the stability of the tetrameric state in Tyr110Phe SDH was decreased as compared to wt SDH where the change in Gibbs energy was (DDG’02) 8 kJ/mol (Table 1). Rattus norvegicus 17952367 1.1.1.14 Y110F 24130 24295 However, the stability of the tetrameric state in Tyr110Phe SDH was decreased as compared to wt SDH where the change in Gibbs energy was (DDG’02) 8 kJ/mol (Table 1). Rattus norvegicus 17952367 1.1.1.14 Y110F 24297 24475 This change in Gibbs energy is equivalent to a 20-fold larger equilibrium constant, which clearly shows that the tetrameric quaternary structure is destabilized in Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 24297 24475 This change in Gibbs energy is equivalent to a 20-fold larger equilibrium constant, which clearly shows that the tetrameric quaternary structure is destabilized in Tyr110Phe SDH. Rattus norvegicus 17952367 1.1.1.14 Y110F 24476 24713 Circular dichroism analysis showed that the overall secondary structure was retained between wt SDH and Tyr110Phe SDH, indicating that the mutation primarily affects the dimer-dimer interaction area and not the subunit structure as such. Rattus norvegicus 17952367 1.1.1.14 Y110F 24476 24713 Circular dichroism analysis showed that the overall secondary structure was retained between wt SDH and Tyr110Phe SDH, indicating that the mutation primarily affects the dimer-dimer interaction area and not the subunit structure as such. Rattus norvegicus 17952367 1.1.1.14 Y110F 29032 29264 In mutagenesis and molecular modeling studies it was shown that the main source for the difference in stability between the bacterial ADHs was due to critical replacements of residues at the interface areas between subunits [6, 25]. Rattus norvegicus 17952367 1.1.1.14 Y110F 31681 31928 Even though a part of Tyr110Phe SDH was eluted as tetramers no enzymatic activity in those fractions was observed, and this proves that not only association between subunits in the tetrameric state is sufficient to induce a catalytic active state. Rattus norvegicus 17952367 1.1.1.14 Y110F 31929 32266 Several site-directed mutagenesis studies have showed that mutations primarily involving changes in the net charge have a significant effect on protein-protein association rates [26, 27], which suggests that the higher stability of wt SDH as compared to Tyr110Phe SDH is probably not caused by a higher rate of association (k2 ; Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 31929 32266 Several site-directed mutagenesis studies have showed that mutations primarily involving changes in the net charge have a significant effect on protein-protein association rates [26, 27], which suggests that the higher stability of wt SDH as compared to Tyr110Phe SDH is probably not caused by a higher rate of association (k2 ; Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 31929 32266 Several site-directed mutagenesis studies have showed that mutations primarily involving changes in the net charge have a significant effect on protein-protein association rates [26, 27], which suggests that the higher stability of wt SDH as compared to Tyr110Phe SDH is probably not caused by a higher rate of association (k2 ; Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 32267 32570 However, electrostatic attraction could still explain part of the change in binding since the mutated residue Tyr110 to Phe destroys the hydrogen-bonding network that links the negatively charged residue Glu94 in one subunit to the positively charged residue Lys106 in another subunit (Table 4, Fig. 5). Rattus norvegicus 17952367 1.1.1.14 Y110F 32571 32769 In line with this we suggest that the decreased stability of the tetrameric state in Tyr110Phe SDH as compared to wt SDH is caused by an increased rate of dissociation (k-2) for the mutant (Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 32571 32769 In line with this we suggest that the decreased stability of the tetrameric state in Tyr110Phe SDH as compared to wt SDH is caused by an increased rate of dissociation (k-2) for the mutant (Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 32571 32769 In line with this we suggest that the decreased stability of the tetrameric state in Tyr110Phe SDH as compared to wt SDH is caused by an increased rate of dissociation (k-2) for the mutant (Fig. 4). Rattus norvegicus 17952367 1.1.1.14 Y110F 32770 32951 The abolished enzymatic function for the tested substrates in Tyr110Phe SDH is probably due to structural changes of the active site due to the altered interaction between subunits. Rattus norvegicus 17952367 1.1.1.14 Y110F 32770 32951 The abolished enzymatic function for the tested substrates in Tyr110Phe SDH is probably due to structural changes of the active site due to the altered interaction between subunits. Rattus norvegicus 17952367 1.1.1.14 Y110F 32952 33088 Most likely these structural changes increase the transition state energy barrier for Tyr110Phe SDH, which destroys its catalytic power. Rattus norvegicus 17952367 1.1.1.14 Y110F 32952 33088 Most likely these structural changes increase the transition state energy barrier for Tyr110Phe SDH, which destroys its catalytic power. Rattus norvegicus 17952367 2.2.1.1 Y440A 1258 1459 This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. Escherichia coli 17969139 2.2.1.1 D381A 1258 1459 This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. Escherichia coli 17969139 2.2.1.1 Y440A 7823 8292 Protein concentration was determined by absorbance at 280 nm, assuming molecular weights and extinction coefficients (Pace et al., 1995), respectively of: 72260.82 g molÀ1 and e ¼ 93,905 MÀ1 cmÀ1 for wild-type; 72216.81 g molÀ1 and e ¼ 93,905 MÀ1 cmÀ1 for D381A; and 72168.72 g molÀ1 and e ¼ 92,415 MÀ1 cmÀ1 for Y440A. Pure transketolase was stored in 250 mM Tris-HCl, pH 7.5, at 48C for a maximum of 2 weeks without loss of activity, and with no precipitation visible. Escherichia coli 17969139 2.2.1.1 D381A 17185 17401 A2 transition, the C1/2 obtained 2 when using a fixed equilibration time at each urea concentration, is still useful for comparing the stability of wild-type under different conditions, or relative to mutant enzymes. Escherichia coli 17969139 2.2.1.1 Y440A 17185 17401 A2 transition, the C1/2 obtained 2 when using a fixed equilibration time at each urea concentration, is still useful for comparing the stability of wild-type under different conditions, or relative to mutant enzymes. Escherichia coli 17969139 2.2.1.1 D381A 18855 19192 Activity Analysis of Wild-Type and Mutant Transketolases Activity measurements, using the enzyme-linked assay on samples with standardized protein concentrations are summarised in Table I. D381A is 56-fold less active than wild-type transketolase, whereas Y440A is even less active with approximately 700-fold less specific activity than Escherichia coli 17969139 2.2.1.1 Y440A 18855 19192 Activity Analysis of Wild-Type and Mutant Transketolases Activity measurements, using the enzyme-linked assay on samples with standardized protein concentrations are summarised in Table I. D381A is 56-fold less active than wild-type transketolase, whereas Y440A is even less active with approximately 700-fold less specific activity than Escherichia coli 17969139 2.2.1.1 Y440A 18855 19192 Activity Analysis of Wild-Type and Mutant Transketolases Activity measurements, using the enzyme-linked assay on samples with standardized protein concentrations are summarised in Table I. D381A is 56-fold less active than wild-type transketolase, whereas Y440A is even less active with approximately 700-fold less specific activity than Escherichia coli 17969139 2.2.1.1 D381A 19909 20099 Previous activity measurements for yeast TK have also shown that the mutant D382A (equivalent to D381A in E. coli) is 43-fold less active than the wild-type enzyme (Meshalkina et al., 1997). Escherichia coli 17969139 2.2.1.1 D381A 20347 20488 D381A shifts the A2 -> U transition midpoint to 4.0 M from 4.7 M urea in the wildtype, suggesting the destabilization of the A2 intermediate. Escherichia coli 17969139 2.2.1.1 D381A 20930 21099 The latter possibility is supported by the 56-fold decrease in activity for D381A and also by the significant redshift in lmax of 5 nm at 0 M urea compared to wild-type. Escherichia coli 17969139 2.2.1.1 D381A 20930 21099 The latter possibility is supported by the 56-fold decrease in activity for D381A and also by the significant redshift in lmax of 5 nm at 0 M urea compared to wild-type. Escherichia coli 17969139 2.2.1.1 D381A 21100 21440 This is also consistent with the apparent loss of TPP affinity observed previously for the equivalent yeast mutant, and the less ordered circulardichroism spectrum under native conditions, which the authors suggested was due to a lower population or altered distribution of catalytic intermediates for this mutant (Meshalkina et al., 1997). Escherichia coli 17969139 2.2.1.1 D381A 21100 21440 This is also consistent with the apparent loss of TPP affinity observed previously for the equivalent yeast mutant, and the less ordered circulardichroism spectrum under native conditions, which the authors suggested was due to a lower population or altered distribution of catalytic intermediates for this mutant (Meshalkina et al., 1997). Escherichia coli 17969139 2.2.1.1 D381A 21100 21440 This is also consistent with the apparent loss of TPP affinity observed previously for the equivalent yeast mutant, and the less ordered circulardichroism spectrum under native conditions, which the authors suggested was due to a lower population or altered distribution of catalytic intermediates for this mutant (Meshalkina et al., 1997). Escherichia coli 17969139 2.2.1.1 D381A 21100 21440 This is also consistent with the apparent loss of TPP affinity observed previously for the equivalent yeast mutant, and the less ordered circulardichroism spectrum under native conditions, which the authors suggested was due to a lower population or altered distribution of catalytic intermediates for this mutant (Meshalkina et al., 1997). Escherichia coli 17969139 2.2.1.1 Y440A 21812 21971 Y440A only marginally destabilizes the A2 intermediate state with an A2 -> U transition midpoint of 4.4 M urea compared to 4.7 M for wild-type (Fig. 4A and B). Escherichia coli 17969139 2.2.1.1 Y440A 22417 22722 This is supported by the 700fold lower activity of Y440A compared to wild-type, although this could also in part result from the altered structure and function of the tunnel between the two TPP cofactors, known to be functionally important for a related enzyme pyruvate dehydrogenase (Frank et al., 2004). Escherichia coli 17969139 2.2.1.1 Y440A 22417 22722 This is supported by the 700fold lower activity of Y440A compared to wild-type, although this could also in part result from the altered structure and function of the tunnel between the two TPP cofactors, known to be functionally important for a related enzyme pyruvate dehydrogenase (Frank et al., 2004). Escherichia coli 17969139 2.2.1.1 Y440A 23845 23924 Assay time of enzyme reactions increase with mutants that display low activity. Escherichia coli 17969139 2.2.1.1 D381A 23845 23924 Assay time of enzyme reactions increase with mutants that display low activity. Escherichia coli 17969139 2.2.1.1 Y440A 23845 23924 Assay time of enzyme reactions increase with mutants that display low activity. Escherichia coli 17969139 2.2.1.1 D381A 23845 23924 Assay time of enzyme reactions increase with mutants that display low activity. Escherichia coli 17969139 2.2.1.1 D381A 25191 25501 Comparison of Wild-Type and Mutant Transketolase Denaturation in Microplates For the increased throughput appropriate for protein stability measurement in future directed evolution or biocatalytic process screening experiments, the serial addition method was used as described previously (Aucamp et al., 2005). Escherichia coli 17969139 2.2.1.1 Y440A 27687 27795 The Y440A mutant displays a lower stability for NÃ relative to wild-type, with the C1/2 2 value for the NÃ ! Escherichia coli 17969139 2.2.1.1 Y440A 29242 29595 Overall, the same ranking of stability was obtained for all the transitions in the 0-4 M urea range, for wild-type, D381A and Y440A. Therefore, this method is suitable for ranking the stabilities of transketolase mutants and also for their intermediates on the unfolding pathway provided they differ in their transition midpoints by at least 0.2 M urea. Escherichia coli 17969139 2.2.1.1 Y440A 29242 29595 Overall, the same ranking of stability was obtained for all the transitions in the 0-4 M urea range, for wild-type, D381A and Y440A. Therefore, this method is suitable for ranking the stabilities of transketolase mutants and also for their intermediates on the unfolding pathway provided they differ in their transition midpoints by at least 0.2 M urea. Escherichia coli 17969139 2.2.1.1 D381A 29242 29595 Overall, the same ranking of stability was obtained for all the transitions in the 0-4 M urea range, for wild-type, D381A and Y440A. Therefore, this method is suitable for ranking the stabilities of transketolase mutants and also for their intermediates on the unfolding pathway provided they differ in their transition midpoints by at least 0.2 M urea. Escherichia coli 17969139 2.2.1.1 D381A 29242 29595 Overall, the same ranking of stability was obtained for all the transitions in the 0-4 M urea range, for wild-type, D381A and Y440A. Therefore, this method is suitable for ranking the stabilities of transketolase mutants and also for their intermediates on the unfolding pathway provided they differ in their transition midpoints by at least 0.2 M urea. Escherichia coli 17969139 2.2.1.1 Y440A 30714 30939 Conclusions D381A and Y440A, show significant destabilization of native and intermediate states of transketolase that can be monitored by changes in the urea denaturation transition mid-points (C1/2) measured by fluorescence. Escherichia coli 17969139 2.2.1.1 D381A 30714 30939 Conclusions D381A and Y440A, show significant destabilization of native and intermediate states of transketolase that can be monitored by changes in the urea denaturation transition mid-points (C1/2) measured by fluorescence. Escherichia coli 17969139 2.2.1.1 Y440A 30940 31086 Loss of stability in the TPP-associated native state was supported by decreased catalytic activities when compared to the wild-type transketolase. Escherichia coli 17969139 2.2.1.1 D381A 30940 31086 Loss of stability in the TPP-associated native state was supported by decreased catalytic activities when compared to the wild-type transketolase. Escherichia coli 17969139 2.2.1.1 Y440A 30940 31086 Loss of stability in the TPP-associated native state was supported by decreased catalytic activities when compared to the wild-type transketolase. Escherichia coli 17969139 2.2.1.1 D381A 30940 31086 Loss of stability in the TPP-associated native state was supported by decreased catalytic activities when compared to the wild-type transketolase. Escherichia coli 17969139 2.2.1.1 Y440A 31856 32112 Transition mid-points can also be determined with sufficient accuracy for resolving altered stabilities of native and unfolding intermediate states, upon mutation or under different biocatalytic process conditions (such as the removal of excess cofactors). Escherichia coli 17969139 2.2.1.1 Y440A 31856 32112 Transition mid-points can also be determined with sufficient accuracy for resolving altered stabilities of native and unfolding intermediate states, upon mutation or under different biocatalytic process conditions (such as the removal of excess cofactors). Escherichia coli 17969139 1.11.1.15 C52S 921 1272 We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. Rattus norvegicus 17974571 1.11.1.15 C52S 921 1272 We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. Rattus norvegicus 17974571 1.11.1.15 C173S 921 1272 We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. Rattus norvegicus 17974571 1.11.1.15 C173S 921 1272 We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. Rattus norvegicus 17974571 1.11.1.15 C83S 1273 1472 The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T. (1999) Proc. Rattus norvegicus 17974571 1.11.1.15 C52S 1517 1661 X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, ∼130Å; inside diameter, ∼55Å; thickness, ∼45Å). Rattus norvegicus 17974571 1.11.1.15 C83S 2204 2338 The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. Rattus norvegicus 17974571 1.11.1.15 C83S 2204 2338 The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. Rattus norvegicus 17974571 1.11.1.15 C83S 4152 4409 We previously reported the crystal structure of the oxidized dimeric form of the rat HBP23/Prx I C83S mutant (PDB entry 1QQ2), in which the active site Cys52 forms a disulfide bridge with Cys173 from another subunit by C-terminal swapping in the dimer (24). Rattus norvegicus 17974571 1.11.1.15 C52S 6862 7702 Here, to address the relationship between the oligomeric properties and peroxidase activity of mammalian 2-Cys Prx, we prepared HBP23/Prx I variants by using site-directed mutagenesis, and we also performed an x-ray structure analysis of decameric Prx I. In contrast to the recently reported result for human Prx I (40), our crystal structure of the decameric rat C52S mutant (Protein Data Bank (PDB) entry 2Z9S) showed that only one out of the five dimer-dimer interfaces involved a disulfide bridge from Cys83 to Cys83 of the adjacent dimer, indicating that stabilization of the decamer structure is not due to Cys83-Cys83 disulfide bridges at the dimer-dimer interfaces, but rather, is predominantly due to hydrophobic interaction and structural fitting, as is seen in the decameric structures of bacterial (26-28) and human (25) Prx II. Rattus norvegicus 17974571 1.11.1.15 C52S 6862 7702 Here, to address the relationship between the oligomeric properties and peroxidase activity of mammalian 2-Cys Prx, we prepared HBP23/Prx I variants by using site-directed mutagenesis, and we also performed an x-ray structure analysis of decameric Prx I. In contrast to the recently reported result for human Prx I (40), our crystal structure of the decameric rat C52S mutant (Protein Data Bank (PDB) entry 2Z9S) showed that only one out of the five dimer-dimer interfaces involved a disulfide bridge from Cys83 to Cys83 of the adjacent dimer, indicating that stabilization of the decamer structure is not due to Cys83-Cys83 disulfide bridges at the dimer-dimer interfaces, but rather, is predominantly due to hydrophobic interaction and structural fitting, as is seen in the decameric structures of bacterial (26-28) and human (25) Prx II. Rattus norvegicus 17974571 1.11.1.15 C52S 6862 7702 Here, to address the relationship between the oligomeric properties and peroxidase activity of mammalian 2-Cys Prx, we prepared HBP23/Prx I variants by using site-directed mutagenesis, and we also performed an x-ray structure analysis of decameric Prx I. In contrast to the recently reported result for human Prx I (40), our crystal structure of the decameric rat C52S mutant (Protein Data Bank (PDB) entry 2Z9S) showed that only one out of the five dimer-dimer interfaces involved a disulfide bridge from Cys83 to Cys83 of the adjacent dimer, indicating that stabilization of the decamer structure is not due to Cys83-Cys83 disulfide bridges at the dimer-dimer interfaces, but rather, is predominantly due to hydrophobic interaction and structural fitting, as is seen in the decameric structures of bacterial (26-28) and human (25) Prx II. Rattus norvegicus 17974571 1.11.1.15 C52S 6862 7702 Here, to address the relationship between the oligomeric properties and peroxidase activity of mammalian 2-Cys Prx, we prepared HBP23/Prx I variants by using site-directed mutagenesis, and we also performed an x-ray structure analysis of decameric Prx I. In contrast to the recently reported result for human Prx I (40), our crystal structure of the decameric rat C52S mutant (Protein Data Bank (PDB) entry 2Z9S) showed that only one out of the five dimer-dimer interfaces involved a disulfide bridge from Cys83 to Cys83 of the adjacent dimer, indicating that stabilization of the decamer structure is not due to Cys83-Cys83 disulfide bridges at the dimer-dimer interfaces, but rather, is predominantly due to hydrophobic interaction and structural fitting, as is seen in the decameric structures of bacterial (26-28) and human (25) Prx II. Rattus norvegicus 17974571 1.11.1.15 C52S 14421 14761 X-ray Crystallography of HBP23/Prx I—The C52S mutant was crystallized by the hanging drop method at 20 °C by mixing 2 μl of the protein solution (5 mg/ml in 5 mm sodium acetate, pH 5.0, 2 mm DTT, 1 mm CHAPS) with 2 μl of reservoir solution (0.17 m ammonium acetate, 20% glycerol, 25% polyethylene glycol 4000, 84 mm sodium acetate, pH 5.0). Rattus norvegicus 17974571 1.11.1.15 C83S 15179 15392 Molecular replacement was performed with EPMR using the coordinates of the crystal structure of human decameric 2-Cys Prx II purified from erythrocytes (Protein Data Bank entry 1QMV; Ref. 22 and see also Ref. 46). Rattus norvegicus 17974571 1.11.1.15 C52S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C173S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C52S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C173S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C52S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C173S 17661 17988 The mutants C52S and C173S, which cannot form an intermolecular disulfide bridge in the vicinity of the active site under oxidative conditions, were eluted only as decamers under all conditions studied (Fig. 2, A and B), which is consistent with the observation that reduced, DTT-treated decameric HBP23/Prx I is rather stable. Rattus norvegicus 17974571 1.11.1.15 C83S/C173S 18082 18286 Upon the addition of DTT, however, the dimer reverted to the monomer rather than forming the decamer, suggesting that the replacement of the sulfur atom with oxygen influences the dimer-dimer interaction. Rattus norvegicus 17974571 1.11.1.15 C52S/C83S 18082 18286 Upon the addition of DTT, however, the dimer reverted to the monomer rather than forming the decamer, suggesting that the replacement of the sulfur atom with oxygen influences the dimer-dimer interaction. Rattus norvegicus 17974571 1.11.1.15 C52S 18620 18844 Crystal Structure of Decameric HBP23/Prx I—To elucidate in detail the structural features of HBP23/Prx I, the C52S mutant was successfully crystallized, and its structure was determined (PDB entry 2Z9S) (supplemental table). Rattus norvegicus 17974571 1.11.1.15 C83S 18845 18920 The final model was compared with that of the oxidized dimeric C83S mutant. Rattus norvegicus 17974571 1.11.1.15 C52S 19381 19518 The C52S mutant was crystallized as a toroidal decamer (Fig. 3) very similar to the reported structures of other Prx species (25-28, 36). Rattus norvegicus 17974571 1.11.1.15 C83S 21537 21682 The most significant difference between the two mutants is the position of the two cysteines (or corresponding Ser52 residue) in the active site. Rattus norvegicus 17974571 1.11.1.15 C52S 21537 21682 The most significant difference between the two mutants is the position of the two cysteines (or corresponding Ser52 residue) in the active site. Rattus norvegicus 17974571 1.11.1.15 C52S/C83S 23539 23746 The wild type and the HBP23/Prx I mutants C83S and C52S/C83S each formed a stable complex with the Trx mutant C36S. However, the HBP23/Prx I mutant C83S/C173S was not trapped on the column (Fig. 6, panel c). Rattus norvegicus 17974571 1.11.1.15 C83S 23539 23746 The wild type and the HBP23/Prx I mutants C83S and C52S/C83S each formed a stable complex with the Trx mutant C36S. However, the HBP23/Prx I mutant C83S/C173S was not trapped on the column (Fig. 6, panel c). Rattus norvegicus 17974571 1.11.1.15 C52S 24427 24645 The finding in this study that the distance between Ser52 and Cys173 in the crystal structure of the C52S mutant (PDB code 2Z9S) is as large as 13 Å suggests that the structure of mutant C52S is catalytically inactive. Rattus norvegicus 17974571 1.11.1.15 C83S 24988 25285 As it was of interest to examine the relationship between decamerization of the wild-type enzyme and peroxidase activity, we compared the reductive half-reaction by reduced Trx at high concentrations of wild-type enzyme (mainly in decameric form) and the C83S mutant (exclusively in dimeric form). Rattus norvegicus 17974571 1.11.1.15 C83S 24988 25285 As it was of interest to examine the relationship between decamerization of the wild-type enzyme and peroxidase activity, we compared the reductive half-reaction by reduced Trx at high concentrations of wild-type enzyme (mainly in decameric form) and the C83S mutant (exclusively in dimeric form). Rattus norvegicus 17974571 1.11.1.15 C83S 24988 25285 As it was of interest to examine the relationship between decamerization of the wild-type enzyme and peroxidase activity, we compared the reductive half-reaction by reduced Trx at high concentrations of wild-type enzyme (mainly in decameric form) and the C83S mutant (exclusively in dimeric form). Rattus norvegicus 17974571 1.11.1.15 C83S 24988 25285 As it was of interest to examine the relationship between decamerization of the wild-type enzyme and peroxidase activity, we compared the reductive half-reaction by reduced Trx at high concentrations of wild-type enzyme (mainly in decameric form) and the C83S mutant (exclusively in dimeric form). Rattus norvegicus 17974571 1.11.1.15 C83S 25769 25909 It is noteworthy that the C83S mutant showed similar activity to that of the wild type in the Trx system under diluted conditions (Table 1). Rattus norvegicus 17974571 1.11.1.15 C83S 26711 27079 The efficiency of time-dependent incorporation of [14C]IAA into the mutant C83S, however, was 1.4-fold higher than that into the wild type during 8-24 min (13 nmol of Trx and 11 nmol of HBP23/Prx I), and then the incorporation reached a plateau level because of consumption of reduced Trx because of simultaneous competitive incorporation of [14C]IAA into reduced Trx. Rattus norvegicus 17974571 1.11.1.15 C83S 26711 27079 The efficiency of time-dependent incorporation of [14C]IAA into the mutant C83S, however, was 1.4-fold higher than that into the wild type during 8-24 min (13 nmol of Trx and 11 nmol of HBP23/Prx I), and then the incorporation reached a plateau level because of consumption of reduced Trx because of simultaneous competitive incorporation of [14C]IAA into reduced Trx. Rattus norvegicus 17974571 1.11.1.15 C83S 26711 27079 The efficiency of time-dependent incorporation of [14C]IAA into the mutant C83S, however, was 1.4-fold higher than that into the wild type during 8-24 min (13 nmol of Trx and 11 nmol of HBP23/Prx I), and then the incorporation reached a plateau level because of consumption of reduced Trx because of simultaneous competitive incorporation of [14C]IAA into reduced Trx. Rattus norvegicus 17974571 1.11.1.15 C83S 27080 27248 The incorporation of radioactivity into Trx was confirmed to be 1.3 times greater with the wild type than with mutant C83S after separation of Trx by means of SDS-PAGE. Rattus norvegicus 17974571 1.11.1.15 C83S 27465 27781 Influence of the Mutation on Peroxidase Activity—To clarify the role of amino acid residues in the active site of HBP23/Prx I, the activity of variants was examined in two ways, i.e. toward H2O2 in the presence of the Trx system, NADPH, Trx, and Trx reductase and toward t-butyl hydroperoxide in the presence of DTT. Rattus norvegicus 17974571 1.11.1.15 C52S 28198 28372 The activity of all mutants determined in the Trx system was not significantly different from that in the DTT system except in the case of mutation at position 173 (Table 1). Rattus norvegicus 17974571 1.11.1.15 C173S 28456 28576 The C173S mutant was fully active using DTT as an electron transfer partner, although it was inactive in the Trx system. Rattus norvegicus 17974571 1.11.1.15 C83S 28719 28912 It should be noted that C83S exhibited activity similar to that of the wild type in the Trx system, and it is present exclusively as the dimer, as determined with size-exclusion chromatography. Rattus norvegicus 17974571 1.11.1.15 C83S/C173S 28913 29177 In addition, the double mutant C83S/C173S exhibited the highest DTT-dependent activity among the variants studied, like the plant enzyme (39), in accordance with the idea that DTT can act in place of Cys173 and that Cys52 is involved in normal peroxidase activity. Rattus norvegicus 17974571 1.11.1.15 C83S 29178 29280 The substitution of the arginine residues at positions 128 and 151 dramatically affected the activity. Rattus norvegicus 17974571 1.11.1.15 C83S 29281 29452 When either of these was replaced with a lysine, alanine, or glutamate residue in the C83S mutant, the activity decreased to 2-7% of that of the wild type in both systems. Rattus norvegicus 17974571 1.11.1.15 C83S 29568 29685 Substitution to lysine at these positions did not restore the activity, probably due to its lower strength as a base. Rattus norvegicus 17974571 1.11.1.15 C83S 29568 29685 Substitution to lysine at these positions did not restore the activity, probably due to its lower strength as a base. Rattus norvegicus 17974571 1.11.1.15 C83S 31752 32051 Although the fact that the mutant C83S cannot form the decamer might support the former possibility, it is more likely that replacement of the sulfur atom by the smaller oxygen atom favors dissociation to the dimer under circumstances where the dimer-dimer interfaces involve only weak interactions. Rattus norvegicus 17974571 1.1.1.145 T177N 1254 1470 The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Homo sapiens 18571493 1.1.1.145 T177N 1254 1470 The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Homo sapiens 18571493 1.1.1.145 T177N 13011 13233 The molecular determinant for the cold inactivation was identified as Thr177 by replacement of this residue in human DHSR4 with Asn, the corresponding residue in pig DHRS4, whose crystal structure has been determined [21]. Homo sapiens 18571493 1.1.1.145 T177N 22642 22965 This mutation significantly improved the instability at 0 °C, although the addition of NADP+ was needed for complete protection against the cold inactivation (Fig. 2), indicating that the difference in residue at position 177 is one of the structural determinants of that in stability between human and other animal DHRS4s. Homo sapiens 18571493 1.1.1.145 T177N 22642 22965 This mutation significantly improved the instability at 0 °C, although the addition of NADP+ was needed for complete protection against the cold inactivation (Fig. 2), indicating that the difference in residue at position 177 is one of the structural determinants of that in stability between human and other animal DHRS4s. Homo sapiens 18571493 1.1.1.145 T177N 22642 22965 This mutation significantly improved the instability at 0 °C, although the addition of NADP+ was needed for complete protection against the cold inactivation (Fig. 2), indicating that the difference in residue at position 177 is one of the structural determinants of that in stability between human and other animal DHRS4s. Homo sapiens 18571493 1.1.1.145 T177N 46625 46721 The observation of significant improvement in cold stability by the T177N mutation clearly indi- Homo sapiens 18571493 1.13.11.52 S167H 14161 14271 In this case, formation of a ferrous-oxy complex is observed, but it is now more unstable than wild-type hIDO. Homo sapiens 19290871 1.13.11.52 S167H 14272 14484 Hence, both S167H variants of hIDO and hTDO form an unstable ferrous-oxy complex, which is in contrast with wild-type hIDO, and suggests that the histidine residue is particularly significant in this regard [12]. Homo sapiens 19290871 1.13.11.52 S167H 14272 14484 Hence, both S167H variants of hIDO and hTDO form an unstable ferrous-oxy complex, which is in contrast with wild-type hIDO, and suggests that the histidine residue is particularly significant in this regard [12]. Homo sapiens 19290871 1.13.11.52 S167H 14485 14801 The very low reduction potential for S167H (−203 mV) is certainly likely to be influential in destabilizing the ferrous-oxy complex, but does not provide a complete explanation because the reduction potential of recombinant hTDO (−92 mV) is higher, and in this enzyme, the ferrous-oxy species is not detected at all. Homo sapiens 19290871 1.13.11.52 S167H 14485 14801 The very low reduction potential for S167H (−203 mV) is certainly likely to be influential in destabilizing the ferrous-oxy complex, but does not provide a complete explanation because the reduction potential of recombinant hTDO (−92 mV) is higher, and in this enzyme, the ferrous-oxy species is not detected at all. Homo sapiens 19290871 1.13.11.52 S167H 14485 14801 The very low reduction potential for S167H (−203 mV) is certainly likely to be influential in destabilizing the ferrous-oxy complex, but does not provide a complete explanation because the reduction potential of recombinant hTDO (−92 mV) is higher, and in this enzyme, the ferrous-oxy species is not detected at all. Homo sapiens 19290871 1.13.11.52 S167H 14485 14801 The very low reduction potential for S167H (−203 mV) is certainly likely to be influential in destabilizing the ferrous-oxy complex, but does not provide a complete explanation because the reduction potential of recombinant hTDO (−92 mV) is higher, and in this enzyme, the ferrous-oxy species is not detected at all. Homo sapiens 19290871 1.1.3.17 H99N 1248 1454 Decreases of 10-fold and 30-fold in the kcat/Km and kcat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on kcat/KO using choline as substrate. Arthrobacter globiformis 19398559 1.1.3.17 H99N 2006 2129 The temperature dependence of the Dkred values suggests disruption of the preorganization in the asparagine variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 9910 10143 The extinction coefficient of the bound flavin in CHO-H99N was determined by concurrently treating free FAD with 10% trichloroacetic acid and subjecting the acidified free FAD to the same experimental conditions as the mutant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 12160 12521 Equal volumes of CHO-H99N and choline (or 1,2-[2H4]choline) were mixed anaerobically in the stopped-flow spectrophotometer resulting in a reaction mixture with a final enzyme concentration of ∼10 μm and substrate concentrations of 0.05 to 10 mm, with each substrate concentration assayed in triplicate (differences in each set of three observed rates were <5%). Arthrobacter globiformis 19398559 1.1.3.17 H99N 12160 12521 Equal volumes of CHO-H99N and choline (or 1,2-[2H4]choline) were mixed anaerobically in the stopped-flow spectrophotometer resulting in a reaction mixture with a final enzyme concentration of ∼10 μm and substrate concentrations of 0.05 to 10 mm, with each substrate concentration assayed in triplicate (differences in each set of three observed rates were <5%). Arthrobacter globiformis 19398559 1.1.3.17 H99N 15174 15500 The choline oxidase variant in which histidine 99, which participates in a covalent linkage with the C8M atom of FAD, was replaced with asparagine was expressed and purified following the same protocol previously described for wild-type choline oxidase (38) with the addition of 10% glycerol to all the purification solutions. Arthrobacter globiformis 19398559 1.1.3.17 H99N 15501 15908 Unlike the wild-type (38, 51, 54) and other choline oxidase variant enzymes (12, 55, 56), which contain a mixture of oxidized flavin and air-stable, anionic flavosemiquinone upon purification, the H99N mutant enzyme was purified with the bound flavin cofactor in the fully oxidized state, as indicated by the UV-visible absorbance spectrum showing absorbance maxima at 390 and 450 nm (supplemental Fig. S1). Arthrobacter globiformis 19398559 1.1.3.17 H99N 16604 16871 Both the appkcat and app(kcat/Km) values, as well as the specific activity of the mutant enzyme, were significantly lower than in the wild-type enzyme, suggesting that protein flavinylation is important for the alcohol oxidation reaction catalyzed by choline oxidase. Arthrobacter globiformis 19398559 1.1.3.17 H99N 17113 17363 The data were best fit with the sequential steady-state kinetic equation (Equation 1), suggesting that replacement of histidine 99 with asparagine does not alter the order of substrate binding and product release with respect to the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 17113 17363 The data were best fit with the sequential steady-state kinetic equation (Equation 1), suggesting that replacement of histidine 99 with asparagine does not alter the order of substrate binding and product release with respect to the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 17364 17577 CHO-H99N catalyzes the oxidation of choline to glycine betaine with a 10-fold decrease in the kcat/Km value, a 30-fold decrease in the kcat value, but with no significant difference in the kcat/KO value (Table 2). Arthrobacter globiformis 19398559 1.1.3.17 H99N 17364 17577 CHO-H99N catalyzes the oxidation of choline to glycine betaine with a 10-fold decrease in the kcat/Km value, a 30-fold decrease in the kcat value, but with no significant difference in the kcat/KO value (Table 2). Arthrobacter globiformis 19398559 1.1.3.17 H99N 17364 17577 CHO-H99N catalyzes the oxidation of choline to glycine betaine with a 10-fold decrease in the kcat/Km value, a 30-fold decrease in the kcat value, but with no significant difference in the kcat/KO value (Table 2). Arthrobacter globiformis 19398559 1.1.3.17 H99N 18070 18321 The kcat/Km and kcat values increased with increasing pH to limiting values at high pH (Fig. 2), consistent with the requirement of an unprotonated group for catalysis in both the reductive half-reaction and the overall turnover of the variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 18070 18321 The kcat/Km and kcat values increased with increasing pH to limiting values at high pH (Fig. 2), consistent with the requirement of an unprotonated group for catalysis in both the reductive half-reaction and the overall turnover of the variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 18070 18321 The kcat/Km and kcat values increased with increasing pH to limiting values at high pH (Fig. 2), consistent with the requirement of an unprotonated group for catalysis in both the reductive half-reaction and the overall turnover of the variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 18656 18900 This value is not significantly different from the pH-independent value of 86,400 m−1 s−1 that was previously reported for the wild-type enzyme (34), consistent with the oxidative half-reaction not being affected in the CHO-H99N variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 18656 18900 This value is not significantly different from the pH-independent value of 86,400 m−1 s−1 that was previously reported for the wild-type enzyme (34), consistent with the oxidative half-reaction not being affected in the CHO-H99N variant enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 20340 20768 The effect of temperature on the rate constant for flavin reduction (kred) and the associate kinetic isotope effects (Dkred) were investigated to probe the hydride transfer reaction catalyzed by CHO-H99N. The rates of flavin reduction were determined under anaerobic conditions at varying concentrations of choline or 1,2-[2H4]choline from 10 to 28 °C in 50 mm sodium pyrophosphate, pH 9, using a stopped-flow spectrophotometer. Arthrobacter globiformis 19398559 1.1.3.17 H99N 20340 20768 The effect of temperature on the rate constant for flavin reduction (kred) and the associate kinetic isotope effects (Dkred) were investigated to probe the hydride transfer reaction catalyzed by CHO-H99N. The rates of flavin reduction were determined under anaerobic conditions at varying concentrations of choline or 1,2-[2H4]choline from 10 to 28 °C in 50 mm sodium pyrophosphate, pH 9, using a stopped-flow spectrophotometer. Arthrobacter globiformis 19398559 1.1.3.17 H99N 22600 23025 All taken together, these thermodynamic data indicate that the hydride transfer reaction in the H99N enzyme is significantly different from that of the wild-type enzyme for which previous results showed a large and temperature-independent kinetic isotope effect on the reductive half-reaction, similar ΔH‡ values for the cleavage of the CH and CD bonds of choline, a negligible ΔEa value, and a large AH′/AD′ ratio (Table 3). Arthrobacter globiformis 19398559 1.1.3.17 H99N 22600 23025 All taken together, these thermodynamic data indicate that the hydride transfer reaction in the H99N enzyme is significantly different from that of the wild-type enzyme for which previous results showed a large and temperature-independent kinetic isotope effect on the reductive half-reaction, similar ΔH‡ values for the cleavage of the CH and CD bonds of choline, a negligible ΔEa value, and a large AH′/AD′ ratio (Table 3). Arthrobacter globiformis 19398559 1.1.3.17 H99N 24113 24730 In common with the wild-type enzyme, the histidine to asparagine variant form of choline oxidase displayed a steady-state kinetic mechanism with oxygen reacting with the reduced flavin before release of the organic product of the reaction, the requirement of an unprotonated group acting as a base in the reductive half-reaction of choline oxidation, the chemical step of CH bond cleavage being rate-limiting in both the reductive half-reaction and the overall enzyme turnover, and the lack of observable ionizable groups that participate in the oxidative half-reaction in which the reduced flavin reacts with oxygen. Arthrobacter globiformis 19398559 1.1.3.17 H99N 24113 24730 In common with the wild-type enzyme, the histidine to asparagine variant form of choline oxidase displayed a steady-state kinetic mechanism with oxygen reacting with the reduced flavin before release of the organic product of the reaction, the requirement of an unprotonated group acting as a base in the reductive half-reaction of choline oxidation, the chemical step of CH bond cleavage being rate-limiting in both the reductive half-reaction and the overall enzyme turnover, and the lack of observable ionizable groups that participate in the oxidative half-reaction in which the reduced flavin reacts with oxygen. Arthrobacter globiformis 19398559 1.1.3.17 H99N 24113 24730 In common with the wild-type enzyme, the histidine to asparagine variant form of choline oxidase displayed a steady-state kinetic mechanism with oxygen reacting with the reduced flavin before release of the organic product of the reaction, the requirement of an unprotonated group acting as a base in the reductive half-reaction of choline oxidation, the chemical step of CH bond cleavage being rate-limiting in both the reductive half-reaction and the overall enzyme turnover, and the lack of observable ionizable groups that participate in the oxidative half-reaction in which the reduced flavin reacts with oxygen. Arthrobacter globiformis 19398559 1.1.3.17 H99N 25051 25507 This conclusion is strongly reinforced by the observation that the second-order rate constant for reaction of the reduced flavin with oxygen (kcat/Koxygen) is essentially the same with values of ∼85,000 m−1 s−1 in the mutant and wild-type enzymes, because one would expect such a critical reaction of flavin oxidation to occur at significantly different rates had the protein integrity of the mutant enzyme been different from that of the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 25051 25507 This conclusion is strongly reinforced by the observation that the second-order rate constant for reaction of the reduced flavin with oxygen (kcat/Koxygen) is essentially the same with values of ∼85,000 m−1 s−1 in the mutant and wild-type enzymes, because one would expect such a critical reaction of flavin oxidation to occur at significantly different rates had the protein integrity of the mutant enzyme been different from that of the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 25051 25507 This conclusion is strongly reinforced by the observation that the second-order rate constant for reaction of the reduced flavin with oxygen (kcat/Koxygen) is essentially the same with values of ∼85,000 m−1 s−1 in the mutant and wild-type enzymes, because one would expect such a critical reaction of flavin oxidation to occur at significantly different rates had the protein integrity of the mutant enzyme been different from that of the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 25051 25507 This conclusion is strongly reinforced by the observation that the second-order rate constant for reaction of the reduced flavin with oxygen (kcat/Koxygen) is essentially the same with values of ∼85,000 m−1 s−1 in the mutant and wild-type enzymes, because one would expect such a critical reaction of flavin oxidation to occur at significantly different rates had the protein integrity of the mutant enzyme been different from that of the wild-type enzyme. Arthrobacter globiformis 19398559 1.1.3.17 H99N 25853 26547 Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (kred) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the kcat/Km and kcat values with choline as substrate, and the lack of significant changes in the kcat/Koxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Arthrobacter globiformis 19398559 1.1.3.17 H99N 25853 26547 Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (kred) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the kcat/Km and kcat values with choline as substrate, and the lack of significant changes in the kcat/Koxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Arthrobacter globiformis 19398559 1.1.3.17 H99N 25853 26547 Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (kred) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the kcat/Km and kcat values with choline as substrate, and the lack of significant changes in the kcat/Koxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Arthrobacter globiformis 19398559 1.1.3.17 H99N 25853 26547 Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (kred) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the kcat/Km and kcat values with choline as substrate, and the lack of significant changes in the kcat/Koxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Arthrobacter globiformis 19398559 1.1.3.17 H99N 25853 26547 Evidence supporting the importance of the covalent linkage to the hydride transfer reaction is the ∼45-fold decrease in the rate constant for anaerobic flavin reduction (kred) with choline as substrate at 25 °C with respect to the wild-type enzyme, along with the 10-fold and 30-fold decreases in the kcat/Km and kcat values with choline as substrate, and the lack of significant changes in the kcat/Koxygen value for the H99N enzyme as compared with the wild-type form of choline oxidase at pH 10 and 25 °C. Similar results were observed upon replacing either one of the other two histidine residues that are located in the active site of choline oxidase, namely His-351 (56) and His-466 (55). Arthrobacter globiformis 19398559 1.1.3.17 H99N 27706 28018 Evidence for this conclusion comes from the comparison of the effects of temperature on the rates of anaerobic flavin reduction (kred) and the related kinetic isotope effects (Dkred) determined with a stopped-flow spectrophotometer for the H99N enzyme (this study) and the wild-type form of choline oxidase (34). Arthrobacter globiformis 19398559 1.1.3.17 H99N 28676 29207 In contrast, the reaction of hydride ion transfer in the H99N enzyme with the flavin non-covalently attached to the protein requires significant sampling of the reactive configuration that is conducive to the tunneling reaction, as shown by the AH′/AD′ ratio not being significantly different from unity, the large value for the ΔEa with protium and deuterium, the effect of temperature on the Dkred values with 1,2-[2H4]choline, and the different ΔH‡ values for the cleavage of the CH and CD bonds of the substrate (63) (Table 3). Arthrobacter globiformis 19398559 1.1.3.17 H99N 28676 29207 In contrast, the reaction of hydride ion transfer in the H99N enzyme with the flavin non-covalently attached to the protein requires significant sampling of the reactive configuration that is conducive to the tunneling reaction, as shown by the AH′/AD′ ratio not being significantly different from unity, the large value for the ΔEa with protium and deuterium, the effect of temperature on the Dkred values with 1,2-[2H4]choline, and the different ΔH‡ values for the cleavage of the CH and CD bonds of the substrate (63) (Table 3). Arthrobacter globiformis 19398559 1.1.3.17 H99N 28676 29207 In contrast, the reaction of hydride ion transfer in the H99N enzyme with the flavin non-covalently attached to the protein requires significant sampling of the reactive configuration that is conducive to the tunneling reaction, as shown by the AH′/AD′ ratio not being significantly different from unity, the large value for the ΔEa with protium and deuterium, the effect of temperature on the Dkred values with 1,2-[2H4]choline, and the different ΔH‡ values for the cleavage of the CH and CD bonds of the substrate (63) (Table 3). Arthrobacter globiformis 19398559 1.1.3.17 H99N 30780 31058 Independent evidence supporting the lack of commitments to catalysis in the Asn-99 variant enzyme is the lack of perturbation in the kinetic pKa value of 8.4 ± 0.1 determined in the pH profile for the kcat/Km value when choline is substituted with 1,2-[2H4]choline as substrate. Arthrobacter globiformis 19398559 1.1.3.17 H99N 31919 32110 In this respect, the H99N enzyme behaves in a fashion similar to that displayed in choline oxidase variants where the other two active site histidine residues were each replaced with alanine. Arthrobacter globiformis 19398559 1.1.3.17 E312Q 1697 1839 In contrast, less than threefold changes in the Kd values for choline were observed in the mutant enzyme with respect to the wild-type enzyme. Arthrobacter globiformis 19653994 1.1.3.17 E312Q 1840 2191 Thus, the conservative substitution of an active site residue that is involved in substrate binding, but not directly in catalysis, affects primarily the kred value that reports on catalysis and minimally the Kd value that reports on binding, thereby providing an example of the interplay that these kinetic parameters may have in enzymatic reactions. Arthrobacter globiformis 19653994 1.1.3.17 E312Q 4416 4556 A recent study on choline oxidase showed a 500-fold increase in the Kd value for choline upon substituting glutamate 312 with glutamine [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 4648 4961 In contrast, the conservative replacement of Glu312 with aspartate, which preserves the negative charge involved in substrate binding, resulted in a slight increase in the binding affinity of the enzyme for the substrate as suggested by the threefold decrease in the Kd value for choline at pH 10.0 and 25 °C [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 4648 4961 In contrast, the conservative replacement of Glu312 with aspartate, which preserves the negative charge involved in substrate binding, resulted in a slight increase in the binding affinity of the enzyme for the substrate as suggested by the threefold decrease in the Kd value for choline at pH 10.0 and 25 °C [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 6189 6657 The mechanistic data presented herein, when compared to the previously published results for the mechanism of hydride transfer in the wild-type enzyme, indicate that in choline oxidase the conservative substitution of a residue involved in substrate binding, but not directly in catalysis, affects primarily the catalytic events associated with the reaction catalyzed by the enzyme with only a minimal effect on the kinetic parameters that report on substrate binding. Arthrobacter globiformis 19653994 1.1.3.17 E312D 6189 6657 The mechanistic data presented herein, when compared to the previously published results for the mechanism of hydride transfer in the wild-type enzyme, indicate that in choline oxidase the conservative substitution of a residue involved in substrate binding, but not directly in catalysis, affects primarily the catalytic events associated with the reaction catalyzed by the enzyme with only a minimal effect on the kinetic parameters that report on substrate binding. Arthrobacter globiformis 19653994 1.1.3.17 E312D 9833 10061 The addition of glucose and glucose oxidase to the enzyme and substrate solutions was required to scavenge trace amounts of oxygen due to the low Km values for oxygen of 62 lM previously determined for the Glu312Asp enzyme1 [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 10777 11212 The effect of temperature on the rate constants for flavin reduction (kred) with choline or 1,2-[2H4]-choline as substrate for the choline oxidase variant Glu312Asp was investigated between 7 and 37 °C. The enzyme (0.01 mM final) was mixed anaerobically with varying concentrations of choline (between 0.05 and 10 mM final) in a stopped-flow spectrophotometer in 50 mM sodium pyrophosphate, pH 8.0, to determine the kred and Kd values. Arthrobacter globiformis 19653994 1.1.3.17 E312D 11623 11830 The lack of pH effects allowed the choice of a pH value at which the mutant enzyme was stable in the anaerobic tonometer for the prolonged times required to perform the stopped-flow experiment, i.e., pH 8.0. Arthrobacter globiformis 19653994 1.1.3.17 E312D 11924 12209 Anaerobic reductions of the Glu312Asp variant of choline oxidase with choline and 1,2-[2H4]-choline at 37 °C. (A) Stopped-flow traces for choline (a) and 1,2-[2H4]-choline (b) at 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 10.0 mM substrate concentrations in 50 mM sodium pyrophosphate, pH 8.0. Arthrobacter globiformis 19653994 1.1.3.17 E312D 12292 12429 B) Rates of flavin reduction in the Glu312Asp enzyme as a function of substrate concentration with choline (d) and 1,2-[2H4]-choline (s). Arthrobacter globiformis 19653994 1.1.3.17 E312D 12716 13087 The averaged observed rate constants for flavin reduction (kobs) with the Glu312Asp enzyme were hyperbolically dependent on the concentration of choline or 1,2-[2H4]-choline (Fig. 1), allowing for the determination of the limiting rate constants for flavin reduction (kred) and the macroscopic equilibrium constants for substrate binding to the enzyme (Kd) using Eq. (2). Arthrobacter globiformis 19653994 1.1.3.17 E312D 12716 13087 The averaged observed rate constants for flavin reduction (kobs) with the Glu312Asp enzyme were hyperbolically dependent on the concentration of choline or 1,2-[2H4]-choline (Fig. 1), allowing for the determination of the limiting rate constants for flavin reduction (kred) and the macroscopic equilibrium constants for substrate binding to the enzyme (Kd) using Eq. (2). Arthrobacter globiformis 19653994 1.1.3.17 E312D 13664 13907 These Kd values are in keeping with the value of 0.1 mM previously determined for the Glu312Asp enzyme at pH 10 and 25 °C [4], which is threefold lower than that determined for the wild-type enzyme under the same conditions (i.e., 0.3 mM)[10]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 13664 13907 These Kd values are in keeping with the value of 0.1 mM previously determined for the Glu312Asp enzyme at pH 10 and 25 °C [4], which is threefold lower than that determined for the wild-type enzyme under the same conditions (i.e., 0.3 mM)[10]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 14991 15194 Similar entropies of activation (ÀTDSà) of $40 kJ molÀ1 were observed with choline and 1,2-[2H4]-choline as substrate for the Glu312Asp enzyme, yielding an isotope effect on the Eyring intercept of $0.9. Arthrobacter globiformis 19653994 1.1.3.17 E312D 14991 15194 Similar entropies of activation (ÀTDSà) of $40 kJ molÀ1 were observed with choline and 1,2-[2H4]-choline as substrate for the Glu312Asp enzyme, yielding an isotope effect on the Eyring intercept of $0.9. Arthrobacter globiformis 19653994 1.1.3.17 E312D 15672 15983 The corresponding difference in the activation energy for the reaction of hydride ion transfer catalyzed by the Glu312Asp enzyme for the cleavage of the C-H and C-D bonds (DEa), which could be estimated from the slope of the line in the Arrhenius plot of the D(kred) values, had a finite value of $6.5 kJ molÀ1. Arthrobacter globiformis 19653994 1.1.3.17 E312D 15672 15983 The corresponding difference in the activation energy for the reaction of hydride ion transfer catalyzed by the Glu312Asp enzyme for the cleavage of the C-H and C-D bonds (DEa), which could be estimated from the slope of the line in the Arrhenius plot of the D(kred) values, had a finite value of $6.5 kJ molÀ1. Arthrobacter globiformis 19653994 1.1.3.17 E312D 19310 19643 In this respect, a previous study showed that the Glu312Asp enzyme maintains the same sequential steady state kinetic mechanism of reaction, identical pKa of 7.5 for the general base that activates choline in catalysis, and similar D(kred) and Kd values with choline as substrate at pH 10.0 and 25 °C as for the wild-type enzyme [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 19310 19643 In this respect, a previous study showed that the Glu312Asp enzyme maintains the same sequential steady state kinetic mechanism of reaction, identical pKa of 7.5 for the general base that activates choline in catalysis, and similar D(kred) and Kd values with choline as substrate at pH 10.0 and 25 °C as for the wild-type enzyme [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 19310 19643 In this respect, a previous study showed that the Glu312Asp enzyme maintains the same sequential steady state kinetic mechanism of reaction, identical pKa of 7.5 for the general base that activates choline in catalysis, and similar D(kred) and Kd values with choline as substrate at pH 10.0 and 25 °C as for the wild-type enzyme [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 19310 19643 In this respect, a previous study showed that the Glu312Asp enzyme maintains the same sequential steady state kinetic mechanism of reaction, identical pKa of 7.5 for the general base that activates choline in catalysis, and similar D(kred) and Kd values with choline as substrate at pH 10.0 and 25 °C as for the wild-type enzyme [4]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 20103 20441 The transfer of the hydride ion in the reaction catalyzed by the Glu312Asp variant enzyme occurs through either environmentally coupled tunneling with significant distance and geometric sampling of the reactive configurations that are compatible with the transfer reaction or classical over-the-barrier transition state without tunneling. Arthrobacter globiformis 19653994 1.1.3.17 E312D 22669 23053 Irrespective of whether the hydride ion in the reaction catalyzed by the Glu312Asp enzyme tunnels with assistance from the environment or is transferred over the energetic barrier that defines the transition state for the reaction, its transfer occurs at a rate that is $200-fold lower than the rate of the reaction in the wild-type enzyme (i.e., at 25 °C 0.4 sÀ1 versus 93 sÀ1) [10]. Arthrobacter globiformis 19653994 1.1.3.17 E312D 23063 23336 The experimentally observed value of 0.5 for the Glu312Asp enzyme is not significantly different from the value of 0.7 that is commonly used to distinguish between environmentally coupled tunneling with geometric and distance sampling and over-the-barrier transition state. Arthrobacter globiformis 19653994 1.1.3.17 E312D 23520 23852 in the wild-type enzyme the hydride ion tunnels from the a-carbon of the substrate to the N(5) atom of the flavin with minimal assistance from the environment, other than the dynamical motions along the reaction coordinate that are compatible with the transfer reaction [7], that is not the case in the Glu312Asp enzyme (see above). Arthrobacter globiformis 19653994 1.1.3.17 E312D 24531 24731 In the Glu312Asp enzyme the same reaction occurs at a rate of 0.4 sÀ1 through either environmentally coupled tunneling within an enzyme-substrate complex that is not preorganized or without tunneling. Arthrobacter globiformis 19653994 1.1.3.17 E312D 24999 25320 Thus, in choline oxidase the conservative substitution of an active site residue that is involved in substrate binding, but not directly in catalysis, has an effect on the kinetic parameters that report on the catalytic events associated with the enzymatic reaction without affecting those reporting on substrate binding. Arthrobacter globiformis 19653994 1.1.1.12 Y318F 64 1158 A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol Lucy Rutten1, Cecile Ribot2,3,4, Blanca Trejo-Aguilar2, Han AB Wösten2 and Ronald P de Vries*2,5 Address: 1Department of Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands, 2Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands, 3Functional Genomics of Plant Pathogenic Fungi UMR 5240 CNRS-UCB-INSA-Bayer CropScience Microbiology, 14-20 Rue Pierre Baizet B.P. 9163, 69263 Lyon cedex 09 France, 4UMR BGPI, Equipe "Interactions riz-parasites", Campus International de Baillarguet, Montpellier, France and 5Fungal Physiology, CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands Email: Lucy Rutten - lucy.rutten@gmail.com; Cecile Ribot - cecile.ribot@supagro.inra.fr; Blanca Trejo-Aguilar - gao@servidor.unam.mx; Han AB Wösten - h.a.b.wosten@uu.nl; Ronald P de Vries* - r.devries@cbs.knaw.nl * Corresponding author Aspergillus niger 19674460 1.1.1.12 Y318F 2670 2849 While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Aspergillus niger 19674460 1.1.1.12 Y318F 2670 2849 While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Aspergillus niger 19674460 1.1.1.12 Y318F 2850 2917 Moreover, the affinity for D-sorbitol was increased in this mutant. Aspergillus niger 19674460 1.1.1.12 Y318F 8149 8415 Analysis of LadA M70F and Y318F Using site directed mutagenesis, specific mutants of LadA were produced in which M70 and Y318 were altered, individually and in combination, to phenylalanine that is present at these positions in xylitol and D-sorbitol dehydrogenases. Aspergillus niger 19674460 1.1.1.12 M70F 8149 8415 Analysis of LadA M70F and Y318F Using site directed mutagenesis, specific mutants of LadA were produced in which M70 and Y318 were altered, individually and in combination, to phenylalanine that is present at these positions in xylitol and D-sorbitol dehydrogenases. Aspergillus niger 19674460 1.1.1.12 M70F 12759 12970 A previous study suggested the presence of a single L-arabitol dehydrogenase encoding gene involved in the L-arabinose catabolism [6], as a UV mutant of this gene was devoid of L-arabitol dehydrogenase activity. Aspergillus niger 19674460 1.1.1.12 M70F 14037 14157 The M70F mutation of LadA of A. niger resulted in almost complete inactivation of the enzyme on a variety of substrates. Aspergillus niger 19674460 1.1.1.12 Y318F 14481 14630 The Y318F mutation of LadA resulted in increased affinity of the enzyme for D-sorbitol, while the Vmax and Kcat increased for L-arabitol and xylitol. Aspergillus niger 19674460 1.1.1.12 Y318F 14481 14630 The Y318F mutation of LadA resulted in increased affinity of the enzyme for D-sorbitol, while the Vmax and Kcat increased for L-arabitol and xylitol. Aspergillus niger 19674460 1.1.1.12 Y318F 14806 14926 This suggests that the OH group on the Y318 is the only structural difference between LadA and the Y318F mutant protein. Aspergillus niger 19674460 1.1.1.12 Y318F 15321 15469 However, the increased activity in the mutant suggests that the presence of the OH-group delays release of the products (L-xylulose and D-xylulose). Aspergillus niger 19674460 1.1.1.12 Y318F 15667 15968 The increased affinity for D-sorbitol but not for L-arabitol or xylitol of the Y318F mutant may suggest that the presence of the OH group on Y318 in LadA interferes with the OH group on C5 of L-arabitol, resulting in a conformation for D-sorbitol in the active site that inhibits enzymatic conversion. Aspergillus niger 19674460 1.1.1.12 Y318F 15667 15968 The increased affinity for D-sorbitol but not for L-arabitol or xylitol of the Y318F mutant may suggest that the presence of the OH group on Y318 in LadA interferes with the OH group on C5 of L-arabitol, resulting in a conformation for D-sorbitol in the active site that inhibits enzymatic conversion. Aspergillus niger 19674460 1.1.1.12 Y318F 16079 16214 Mutations that result in beneficial changes are likely to be maintained, while disadvantageous mutations will lose ND = not determined. Aspergillus niger 19674460 1.1.1.12 Y318F 16079 16214 Mutations that result in beneficial changes are likely to be maintained, while disadvantageous mutations will lose ND = not determined. Aspergillus niger 19674460 1.1.1.12 Y318F 16318 16508 kinetic properties of wild type LadA and the Y318F mutant protein demonstrated that the Y318F mutant protein had a higher Vmax on L-arabitol and xylitol, but similar affinity (Km) (Table 2). Aspergillus niger 19674460 1.1.1.12 Y318F 16318 16508 kinetic properties of wild type LadA and the Y318F mutant protein demonstrated that the Y318F mutant protein had a higher Vmax on L-arabitol and xylitol, but similar affinity (Km) (Table 2). Aspergillus niger 19674460 1.1.1.12 Y318F 16509 16655 In contrast, the Vmax on Dsorbitol was similar for LadA and the Y318F mutant protein, but the Km of the mutant was nearly 5-times lower (Table 2). Aspergillus niger 19674460 1.1.1.12 Y318F 16509 16655 In contrast, the Vmax on Dsorbitol was similar for LadA and the Y318F mutant protein, but the Km of the mutant was nearly 5-times lower (Table 2). Aspergillus niger 19674460 1.1.1.12 Y318F 18498 18712 The higher activity on L-arabitol of the Y318F mutant protein suggests an evolutionary advantage for this mutation with respect to conversion of this compound and therefore the efficiency of this metabolic pathway. Aspergillus niger 19674460 1.1.1.12 Y318F 18860 19000 Alternatively, since the increased activity is accompanied by a reduction in specificity this could provide selection against this mutation. Aspergillus niger 19674460 1.1.1.12 Y318F 18860 19000 Alternatively, since the increased activity is accompanied by a reduction in specificity this could provide selection against this mutation. Aspergillus niger 19674460 1.2.1.2 Q313E 1173 1391 The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gin 313 is essential for the broad pH affinity profile towards substrate. Pseudomonas sp. 8706817 1.2.1.2 Q313E 1173 1391 The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gin 313 is essential for the broad pH affinity profile towards substrate. Pseudomonas sp. 8706817 1.2.1.2 H332F 1392 1455 His332phe mutation leads to a complete loss of enzyme activity. Pseudomonas sp. 8706817 1.2.1.2 H332F 8564 8661 The concentration of inactive His332Phe mutant enzyme was measured by ELISA as described in [15]. Pseudomonas sp. 8706817 1.2.1.2 H332F 10360 10475 Mutation of His 332 to any residue without the ability to form H-bonds is thus expected to destroy formate binding. Pseudomonas sp. 8706817 1.2.1.2 H332F 10874 10970 The His332Phe mutant has no catalytic activity, attributed to loss of ability to bind substrate. Pseudomonas sp. 8706817 1.2.1.2 H332F 11252 11416 According to experiments on quenching of protein fluorescence by coenzyme (Table 1), the His332Phe mutant has nearly the same affinity for N A D + as wild-type FDH. Pseudomonas sp. 8706817 1.2.1.2 Q313E 11652 11747 The mutant has the same catalytic activity as the wild type and the same affinity for coenzyme. Pseudomonas sp. 8706817 1.2.1.2 Q313E 11652 11747 The mutant has the same catalytic activity as the wild type and the same affinity for coenzyme. Pseudomonas sp. 8706817 1.2.1.2 Q313E 11748 11895 The mutant showed stronger formate binding than the wild type in the pH range 6.0-7.0 where His 332 is expected to be protonated (Table 1, Fig. 4). Pseudomonas sp. 8706817 1.2.1.2 Q313E 12682 12796 Thus, the Gln313Glu mutation affects the binding of formate to the active site due to change of the pK of His 332. Pseudomonas sp. 8706817 1.2.1.2 Q313E 12900 13013 Both the wild type and the Gln313Glu mutant show the same catalytic activity within a wide range of pHs (Fig. 4). Pseudomonas sp. 8706817 1.2.1.2 Q313E 13275 13417 The affinity of the Gln313Glu mutant towards substrates is slightly superior to the wild type at neutral pH but rapidly decreases at basic pH. Pseudomonas sp. 8706817 1.1.5.2 H775A 746 922 Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 H775R 746 922 Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 H775R 746 922 Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 H775A 746 922 Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 H775R 923 1091 H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His775 may directly interact with PQQ. Escherichia coli 9705344 1.1.5.2 H775A 923 1091 H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His775 may directly interact with PQQ. Escherichia coli 9705344 1.1.5.2 D730A 1092 1249 D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2؉, or substrate, but D730R showed reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730N 1092 1249 D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2؉, or substrate, but D730R showed reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730R 1092 1249 D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2؉, or substrate, but D730R showed reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730N 1092 1249 D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2؉, or substrate, but D730R showed reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730A 1092 1249 D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2؉, or substrate, but D730R showed reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730N 1524 1710 Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Escherichia coli 9705344 1.1.5.2 D730R 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 G689A 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 R687A 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 H775A 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 R687D 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 D730A 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 D693A 6757 6948 To avoid mutants producing immature GDHs, the isolates were then examined by Western blot using an antibody raised against GDH as described previously (35) and by measurement of GDH activity. Escherichia coli 9705344 1.1.5.2 G689D 10180 10342 Purification of Mutant GDHs—Four mutant GDHs with significant effect on the enzyme activity were purified according to the procedure as described previously (10). Escherichia coli 9705344 1.1.5.2 S357L 10180 10342 Purification of Mutant GDHs—Four mutant GDHs with significant effect on the enzyme activity were purified according to the procedure as described previously (10). Escherichia coli 9705344 1.1.5.2 G689D 10343 10459 In S357L and G689D, all purification steps were performed in the presence of 10 nM PQQ to stabilize enzyme activity. Escherichia coli 9705344 1.1.5.2 S357L 10343 10459 In S357L and G689D, all purification steps were performed in the presence of 10 nM PQQ to stabilize enzyme activity. Escherichia coli 9705344 1.1.5.2 S357L 11179 11287 The mutation effect of some mutants were further examined by introducing different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 G689D 11179 11287 The mutation effect of some mutants were further examined by introducing different amino acid substitutions. Escherichia coli 9705344 1.1.5.2 S357L 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 G689D 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 S357L 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 G689D 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 S357L 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 G689D 12732 13057 Subcloning the gcd Gene into a Low Copy Plasmid—To certainly obtain gcd mutants, the gcd gene on a high copy plasmid, pUCGCD1 (19), was subcloned into a low copy plasmid of a pACYC177 derivative, pACYC177-322, in which the large BamHI-PstI fragment of pACYC177 had been connected with the small BamHI-PstI fragment of pBR322. Escherichia coli 9705344 1.1.5.2 H775R 15753 15972 Isolation of the gcd Mutants—Out of 30 GcdϪ mutants isolated from region-specific mutagenesis, targeting the conserved C terminus of GDH, four were found to exhibit less than 10% PMS reductase activity of the wild type. Escherichia coli 9705344 1.1.5.2 H775A 19038 19211 pACGCD2M5 has a nonsense mutation at the 765th codon in the gcd gene, resulting in producing an immature GDH presumably susceptible to intracellular proteolytic degradation. Escherichia coli 9705344 1.1.5.2 G741S 19212 19439 The mutated protein from pACGCD2M9 is expected to have an extra 18 amino acid residues because of mutation of the intrinsic stop codon, which was indicated as a slightly slow migration in SDS-polyacrylamide gel electrophoresis. Escherichia coli 9705344 1.1.5.2 G741S 19440 19567 Out of 32 GcdϪ mutants isolated from hydroxylamine mutagenesis, four had less than 10% PMS reductase activity of the wild type. Escherichia coli 9705344 1.1.5.2 G741S 20520 20739 The results revealed that all mutant GDHs have PMS reductase activity less than 10% of the wild type, with comparable Q-1 reductase and glucose oxidase activities except for G741S and -797K (where - means a stop codon). Escherichia coli 9705344 1.1.5.2 G741S 20740 20983 The latter two mutant GDHs seem to retain equivalent activities of PMS reductase and glucose oxidase to those of the wild type, so their apparent reduced activities may be due to lower content of GDH in the membrane than that of the wild type. Escherichia coli 9705344 1.1.5.2 G741S 20740 20983 The latter two mutant GDHs seem to retain equivalent activities of PMS reductase and glucose oxidase to those of the wild type, so their apparent reduced activities may be due to lower content of GDH in the membrane than that of the wild type. Escherichia coli 9705344 1.1.5.2 G741S 20740 20983 The latter two mutant GDHs seem to retain equivalent activities of PMS reductase and glucose oxidase to those of the wild type, so their apparent reduced activities may be due to lower content of GDH in the membrane than that of the wild type. Escherichia coli 9705344 1.1.5.2 G741S 20740 20983 The latter two mutant GDHs seem to retain equivalent activities of PMS reductase and glucose oxidase to those of the wild type, so their apparent reduced activities may be due to lower content of GDH in the membrane than that of the wild type. Escherichia coli 9705344 1.1.5.2 G741S 20984 21218 The mutant -797K reduced Q-1 reductase activity without the significant effect on Km values for PQQ and Mg2ϩ or on glucose oxidase activity, which reflects the normal electron transfer from GDH via intrinsic Q-8 to cytochrome oxidase. Escherichia coli 9705344 1.1.5.2 G741S 20984 21218 The mutant -797K reduced Q-1 reductase activity without the significant effect on Km values for PQQ and Mg2ϩ or on glucose oxidase activity, which reflects the normal electron transfer from GDH via intrinsic Q-8 to cytochrome oxidase. Escherichia coli 9705344 1.1.5.2 H775R 21219 21432 Thus, it seems that the additional C-terminal 18 amino acid residues of the mutant GDH may hamper access of the artificial Q-1, or change the conformation of the Q-1 reacting site but not of the Q-8 reacting site. Escherichia coli 9705344 1.1.5.2 G689D 21219 21432 Thus, it seems that the additional C-terminal 18 amino acid residues of the mutant GDH may hamper access of the artificial Q-1, or change the conformation of the Q-1 reacting site but not of the Q-8 reacting site. Escherichia coli 9705344 1.1.5.2 E742G/P757L 21219 21432 Thus, it seems that the additional C-terminal 18 amino acid residues of the mutant GDH may hamper access of the artificial Q-1, or change the conformation of the Q-1 reacting site but not of the Q-8 reacting site. Escherichia coli 9705344 1.1.5.2 H775R 23953 24101 S357L, G689D, and H775R showed significantly increased Km values for PQQ, and especially, H775R had 230fold higher Km values for PQQ than wild type. Escherichia coli 9705344 1.1.5.2 S357L 23953 24101 S357L, G689D, and H775R showed significantly increased Km values for PQQ, and especially, H775R had 230fold higher Km values for PQQ than wild type. Escherichia coli 9705344 1.1.5.2 H775R 23953 24101 S357L, G689D, and H775R showed significantly increased Km values for PQQ, and especially, H775R had 230fold higher Km values for PQQ than wild type. Escherichia coli 9705344 1.1.5.2 G689D 23953 24101 S357L, G689D, and H775R showed significantly increased Km values for PQQ, and especially, H775R had 230fold higher Km values for PQQ than wild type. Escherichia coli 9705344 1.1.5.2 E742G/P757L 24102 24199 P326L, G689D, G741S, and the double mutant E742G/P757L showed slightly higher Km values for Mg2ϩ. Escherichia coli 9705344 1.1.5.2 E742G/P757L 24200 24349 All showed nearly the same Km value for glucose as that of wild type, indicating that no mutant appears to be affected at the substrate-binding site. Escherichia coli 9705344 1.1.5.2 E742G/P757L 24200 24349 All showed nearly the same Km value for glucose as that of wild type, indicating that no mutant appears to be affected at the substrate-binding site. Escherichia coli 9705344 1.1.5.2 E742G/P757L 24350 24579 Since GDH has some activities for fucose, galactose, xylose, mannose, and maltose (39), substrate specificity of the mutant GDHs was tested but no mutant showed different substrate specificity from the wild type (data not shown). Escherichia coli 9705344 1.1.5.2 S357L 24580 24787 Therefore, it is likely that no mutation occurs in amino acid residues related to the substrate binding, and that the mutations in these mutant GDHs may not cause total conformational changes of the protein. Escherichia coli 9705344 1.1.5.2 H775R 24580 24787 Therefore, it is likely that no mutation occurs in amino acid residues related to the substrate binding, and that the mutations in these mutant GDHs may not cause total conformational changes of the protein. Escherichia coli 9705344 1.1.5.2 G689D 24580 24787 Therefore, it is likely that no mutation occurs in amino acid residues related to the substrate binding, and that the mutations in these mutant GDHs may not cause total conformational changes of the protein. Escherichia coli 9705344 1.1.5.2 G689D 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 H775R 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 G689D 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 S357L 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 H775R 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 S357L 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 D730N 24788 25090 Of those, four significant and intriguing mutant GDHs were subjected to purification for further characterization, in which S357L, G689D, and H775R showed much lower affinity for PQQ and D730N had reduced activities of PMS reductase and glucose oxidase but normal affinities for PQQ, Mg2ϩ, and glucose. Escherichia coli 9705344 1.1.5.2 G689D 25091 25402 Both S357L and G689D were purified only when PQQ was added to solutions used in purification steps, suggesting that Ser-357 and Gly-689 are located near the PQQ-binding site and both mutations lead to a release of PQQ from the enzyme which may cause deleterious conformational change of GDH during purification. Escherichia coli 9705344 1.1.5.2 S357L 25091 25402 Both S357L and G689D were purified only when PQQ was added to solutions used in purification steps, suggesting that Ser-357 and Gly-689 are located near the PQQ-binding site and both mutations lead to a release of PQQ from the enzyme which may cause deleterious conformational change of GDH during purification. Escherichia coli 9705344 1.1.5.2 D730N 25672 25891 All four purified mutant GDHs showed Vmax less than 5% of the wild type and equivalent affinities for glucose and Mg2ϩ to those of the wild type, and all except for D730N were confirmed to have reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 D730N 25672 25891 All four purified mutant GDHs showed Vmax less than 5% of the wild type and equivalent affinities for glucose and Mg2ϩ to those of the wild type, and all except for D730N were confirmed to have reduced affinity for PQQ. Escherichia coli 9705344 1.1.5.2 S357L 26331 26497 Mutations Influencing on Affinity for PQQ—Among the mutant GDHs, the altered amino acid residues of S357L and H775R are located close to the active site in the model. Escherichia coli 9705344 1.1.5.2 H775R 26331 26497 Mutations Influencing on Affinity for PQQ—Among the mutant GDHs, the altered amino acid residues of S357L and H775R are located close to the active site in the model. Escherichia coli 9705344 1.1.5.2 S357L 27084 27249 S357L mutation influenced the affinity for PQQ but not for Mg2ϩ, although Thr-353 beside Ser-357 hydrogen bonds to PQQ, and Asp-354 and Asn-355 to Ca2ϩ in the model. Escherichia coli 9705344 1.1.5.2 S357L 27250 27500 From these results and the evidence that S357L purification required the addition of PQQ, it is suggested that the Ser to Leu mutation in S357L may cause a local structural change and also influence the position of Thr-353 to reduce affinity for PQQ. Escherichia coli 9705344 1.1.5.2 S357L 27250 27500 From these results and the evidence that S357L purification required the addition of PQQ, it is suggested that the Ser to Leu mutation in S357L may cause a local structural change and also influence the position of Thr-353 to reduce affinity for PQQ. Escherichia coli 9705344 1.1.5.2 H775R 27501 27565 H775R showed an extremely reduced affinity for PQQ and low Vmax. Escherichia coli 9705344 1.1.5.2 H775R 27501 27565 H775R showed an extremely reduced affinity for PQQ and low Vmax. Escherichia coli 9705344 1.1.5.2 H775A 27566 27744 To test whether there is a side effect by the alteration from His to Arg or not, we constructed a mutant H775A with substitution of a relatively small residue (Table IV, Fig. 3). Escherichia coli 9705344 1.1.5.2 H775A 27745 27862 The mutant also showed a low affinity for PQQ, but had an equivalent PMS reductase activity to that of the wild type. Escherichia coli 9705344 1.1.5.2 H775A 27863 28053 These results suggest that His-775 may directly interact with PQQ and that the Arg mutation but not the Ala mutation may disturb the conformation of the active site to decrease the turnover. Escherichia coli 9705344 1.1.5.2 H775N 28054 28257 We assume that His-775 hydrogen bonds to the C2 carboxyl group of PQQ instead of Ser-777 proposed in the model and the His-775 substitution with Arg or Ala gave rise to reduction in the affinity for PQQ. Escherichia coli 9705344 1.1.5.2 H775R 28793 29057 Our experiments revealed that the Km values for maltose of H775R and H775A were 150 and 53 mM, respectively, which were slightly lower than that of the wild type GDH, being 250 mM, although their Km values for glucose were nearly the same as that of the wild type. Escherichia coli 9705344 1.1.5.2 H775A 28793 29057 Our experiments revealed that the Km values for maltose of H775R and H775A were 150 and 53 mM, respectively, which were slightly lower than that of the wild type GDH, being 250 mM, although their Km values for glucose were nearly the same as that of the wild type. Escherichia coli 9705344 1.1.5.2 H775R 28793 29057 Our experiments revealed that the Km values for maltose of H775R and H775A were 150 and 53 mM, respectively, which were slightly lower than that of the wild type GDH, being 250 mM, although their Km values for glucose were nearly the same as that of the wild type. Escherichia coli 9705344 1.1.5.2 H775A 28793 29057 Our experiments revealed that the Km values for maltose of H775R and H775A were 150 and 53 mM, respectively, which were slightly lower than that of the wild type GDH, being 250 mM, although their Km values for glucose were nearly the same as that of the wild type. Escherichia coli 9705344 1.1.5.2 G689D 30091 30269 discrepancy between the mutants of G. oxydans and E. coli might be due to difference of the substituted residues or of residues around the His residue in each tertiary structure. Escherichia coli 9705344 1.1.5.2 G689A 30404 30528 The affinity of G689A was similar to that of wild type although its PMS reductase activity was lower than that of wild type. Escherichia coli 9705344 1.1.5.2 G689A 30404 30528 The affinity of G689A was similar to that of wild type although its PMS reductase activity was lower than that of wild type. Escherichia coli 9705344 1.1.5.2 G689A 30529 30738 Gly residues often form restrictive bond angles, so that the Gly substitution with other amino acid residues may perturb the local structure and adjacent residues that may have a functional role will be moved. Escherichia coli 9705344 1.1.5.2 R687D 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 R687A 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 R687A 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 D693A 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 D693A 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 R687D 30739 31064 Thus, assuming that a residue(s) close to Gly-689 may be involved in binding to PQQ, we targeted two conserved charged amino acid residues, Arg687 and Asp-693, near Gly-689 to construct three mutants, R687A, R687D, and D693A. The former two mutant proteins were found to be low in membrane fractions and seems to be unstable. Escherichia coli 9705344 1.1.5.2 G689A 31369 31631 Because Arg-687 occurs on the outer ␤-sheet, D-strand, of W6 ␤-sheets (27) and its substitutions seemed to enhance degradation of GDH, the residue is assumed to stabilize the superbarrel structure presumably via ionic interaction to a residue on another ␤-sheet. Escherichia coli 9705344 1.1.5.2 G689D 31369 31631 Because Arg-687 occurs on the outer ␤-sheet, D-strand, of W6 ␤-sheets (27) and its substitutions seemed to enhance degradation of GDH, the residue is assumed to stabilize the superbarrel structure presumably via ionic interaction to a residue on another ␤-sheet. Escherichia coli 9705344 1.1.5.2 G689A 31369 31631 Because Arg-687 occurs on the outer ␤-sheet, D-strand, of W6 ␤-sheets (27) and its substitutions seemed to enhance degradation of GDH, the residue is assumed to stabilize the superbarrel structure presumably via ionic interaction to a residue on another ␤-sheet. Escherichia coli 9705344 1.1.5.2 G689D 31369 31631 Because Arg-687 occurs on the outer ␤-sheet, D-strand, of W6 ␤-sheets (27) and its substitutions seemed to enhance degradation of GDH, the residue is assumed to stabilize the superbarrel structure presumably via ionic interaction to a residue on another ␤-sheet. Escherichia coli 9705344 1.1.5.2 G689D 31632 31785 These mutation effects appear to be different from that of G689D or G689A because G689D in the presence of PQQ and G689A even in the absence were stable. Escherichia coli 9705344 1.1.5.2 G689A 31632 31785 These mutation effects appear to be different from that of G689D or G689A because G689D in the presence of PQQ and G689A even in the absence were stable. Escherichia coli 9705344 1.1.5.2 G689A 31632 31785 These mutation effects appear to be different from that of G689D or G689A because G689D in the presence of PQQ and G689A even in the absence were stable. Escherichia coli 9705344 1.1.5.2 G689D 34904 35333 the possibility that around Gly-689 there are specific residue(s) interacting with PQQ or contributing to catalytic function, these data together with those of G698D and G689A suggest that the substituted Asp residue of G689D caused reduction of the affinity for PQQ presumably by changing the confomation around the PQQ-binding site, and that the Gly-689 in the wild type would give a crucial local structure around the residue. Escherichia coli 9705344 1.1.5.2 P326L 35752 35896 Pro-326 occurs in the loop f of the model which is absent from MDH and P326L influenced on affinity for Mg2ϩ more than other mutants (Table II). Escherichia coli 9705344 1.1.5.2 D730N 36395 36673 Both mutations as well as -797K, which has the longer C terminus that may disturb the W8 structure, reduced the content of the mutant proteins in the membrane fractions (Fig. 2 and Table II), suggesting that the conserved region is crucial for stability of the enzyme structure. Escherichia coli 9705344 1.1.5.2 D730N 36395 36673 Both mutations as well as -797K, which has the longer C terminus that may disturb the W8 structure, reduced the content of the mutant proteins in the membrane fractions (Fig. 2 and Table II), suggesting that the conserved region is crucial for stability of the enzyme structure. Escherichia coli 9705344 1.1.5.2 D730N 36834 37028 Possible Function of Asp-730 —D730N showed significantly decreased activities of PMS reductase, Q-1 reductase, and glucose oxidase, but no effect on affinity for PQQ or Mg2ϩ (Tables II and III). Escherichia coli 9705344 1.1.5.2 D730R 37029 37115 The substitution from Asp to Asn does not seem to cause a large conformational change. Escherichia coli 9705344 1.1.5.2 D730A 37029 37115 The substitution from Asp to Asn does not seem to cause a large conformational change. Escherichia coli 9705344 1.1.5.2 D730N 37270 37667 Both mutants also showed low PMS reductase activities comparable to D730N. In contrast to D730N and D730A, Km for PQQ was increased in D730R. Thus, to define whether or not the D730N mutation affects the local structure around the PQQ-binding site, we examined the local conformational change by measuring quenching of intrinsic tryptophan fluorescence induced by addition of PQQ (data not shown). Escherichia coli 9705344 1.1.5.2 D730N 37270 37667 Both mutants also showed low PMS reductase activities comparable to D730N. In contrast to D730N and D730A, Km for PQQ was increased in D730R. Thus, to define whether or not the D730N mutation affects the local structure around the PQQ-binding site, we examined the local conformational change by measuring quenching of intrinsic tryptophan fluorescence induced by addition of PQQ (data not shown). Escherichia coli 9705344 1.1.5.2 D730N 37270 37667 Both mutants also showed low PMS reductase activities comparable to D730N. In contrast to D730N and D730A, Km for PQQ was increased in D730R. Thus, to define whether or not the D730N mutation affects the local structure around the PQQ-binding site, we examined the local conformational change by measuring quenching of intrinsic tryptophan fluorescence induced by addition of PQQ (data not shown). Escherichia coli 9705344 1.1.5.2 D730R 37270 37667 Both mutants also showed low PMS reductase activities comparable to D730N. In contrast to D730N and D730A, Km for PQQ was increased in D730R. Thus, to define whether or not the D730N mutation affects the local structure around the PQQ-binding site, we examined the local conformational change by measuring quenching of intrinsic tryptophan fluorescence induced by addition of PQQ (data not shown). Escherichia coli 9705344 1.1.5.2 D730N 37668 37817 Both purified apo-GDHs of D730N and the wild type exhibited the same fluorescence spectrum with an emission maximum at 338 nm when excited at 280 nm. Escherichia coli 9705344 1.1.5.2 N730D 37930 38167 These results suggest that the Asp to Asn substitution at position 730 may not change the local conformation surrounding PQQ but the Arg substitution may influence the structure around the PQQ-binding site to reduce the affinity for PQQ. Escherichia coli 9705344 1.1.5.2 N730D 37930 38167 These results suggest that the Asp to Asn substitution at position 730 may not change the local conformation surrounding PQQ but the Arg substitution may influence the structure around the PQQ-binding site to reduce the affinity for PQQ. Escherichia coli 9705344 1.1.5.2 N730D 38293 38501 Although the large difference in conformational effect was observed among Asp-730 mutants, all of them exhibited largely decreased PMS reductase activity, suggesting that Asp730 may have a catalytic function. Escherichia coli 9705344 1.1.5.2 N730D 38293 38501 Although the large difference in conformational effect was observed among Asp-730 mutants, all of them exhibited largely decreased PMS reductase activity, suggesting that Asp730 may have a catalytic function. Escherichia coli 9705344 1.1.1.103 C38S 1143 1250 The circular dichroism spectra of these mutants were essentially identical to that of the wild-type enzyme. Escherichia coli K-12 9784233 1.1.1.103 C38D 1251 1387 Mutant C38S was catalytically inactive but mutant C38D had a specific activity of 0.2 unit/mg, a level ~1% that of the wild-type enzyme. Escherichia coli K-12 9784233 1.1.1.103 C38D 1551 1719 Preincubation of mutant C38D with 5 mM Zn2+, Co2+, or Cd2+ increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+ halved and 0.5 mM Hg2+ abolished activity. Escherichia coli K-12 9784233 1.1.1.103 C38D 1551 1719 Preincubation of mutant C38D with 5 mM Zn2+, Co2+, or Cd2+ increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+ halved and 0.5 mM Hg2+ abolished activity. Escherichia coli K-12 9784233 1.1.1.103 C38D 1720 1807 Zn2+-stimulated mutant C38D showed these properties: apparent substrate actiL-Threonine Escherichia coli K-12 9784233 1.1.1.103 C38D 2055 2260 Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 2-amino3-hydroxypentanoate. Escherichia coli K-12 9784233 1.1.1.103 C38D 2055 2260 Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 2-amino3-hydroxypentanoate. Escherichia coli K-12 9784233 1.1.1.103 C38S 5674 5966 The observation that inactivation of E. coli TDH by N-ethyl-5-phenylisoxazolium-3Ј-sulfonate (Woodward’s reagent K) is also due to modification of Cys-38 (9) and the recent isolation of an inactive Tyr-38 mutant of this enzyme (10) further reinforce the importance of this amino acid residue. Escherichia coli K-12 9784233 1.1.1.103 C38S 5674 5966 The observation that inactivation of E. coli TDH by N-ethyl-5-phenylisoxazolium-3Ј-sulfonate (Woodward’s reagent K) is also due to modification of Cys-38 (9) and the recent isolation of an inactive Tyr-38 mutant of this enzyme (10) further reinforce the importance of this amino acid residue. Escherichia coli K-12 9784233 1.1.1.103 C38S 7218 7518 Site-directed mutagenesis experiments with E. coli TDH described in this report represent first attempts to probe the role of Cys-38 in TDH activity, the cause for inactivation of TDH by chemical modification of this residue, and the lack of any detectable activity present in a Tyr-38 mutant of TDH. Escherichia coli K-12 9784233 1.1.1.103 C38S 7218 7518 Site-directed mutagenesis experiments with E. coli TDH described in this report represent first attempts to probe the role of Cys-38 in TDH activity, the cause for inactivation of TDH by chemical modification of this residue, and the lack of any detectable activity present in a Tyr-38 mutant of TDH. Escherichia coli K-12 9784233 1.1.1.103 C38S 7519 7771 After the conservative TDH mutant C38S was found to be catalytically inactive, we mutated Cys-38 to the negatively charged aspartate and glutamate residues on the premise they might ligate a catalytic metal ion and provide a substrate recognition site. Escherichia coli K-12 9784233 1.1.1.103 C38E 12966 13198 The DNA sequence obtained for pYC38S, containing the mutation of Cys-38 (5Ј-CATCTGC-3Ј) to Ser (5Ј- GAT AT CA-3Ј), confirmed that the underlined nucleotides were the only ones altered and that Cys-38 was the sole amino acid changed. Escherichia coli K-12 9784233 1.1.1.103 C38D 12966 13198 The DNA sequence obtained for pYC38S, containing the mutation of Cys-38 (5Ј-CATCTGC-3Ј) to Ser (5Ј- GAT AT CA-3Ј), confirmed that the underlined nucleotides were the only ones altered and that Cys-38 was the sole amino acid changed. Escherichia coli K-12 9784233 1.1.1.103 C38S 15444 15666 Purification of mutant C38S. Although mutant C38S was catalytically inactive, the procedure used to obtain wild-type TDH in homogeneous form was used to isolate and purify this mutant from SP1192/pYC38S cell-free extracts. Escherichia coli K-12 9784233 1.1.1.103 C38S 16031 16161 The purity of mutant C38S was assessed by staining with Coomassie blue after carrying out SDS-PAGE under reducing conditions (26). Escherichia coli K-12 9784233 1.1.1.103 C38D 16925 17004 The purity of mutant C38D was determined by SDS-PAGE under reducing conditions. Escherichia coli K-12 9784233 1.1.1.103 C38D 23245 23541 Determination of the substrate specificity of native as well as metal ion-activated wild-type TDH and mutant C38D. The rate of oxidation of various substrates by wild-type TDH which had been incubated on ice for 5 min alone or with either 0.5 mM Cd2ϩ or 1 mM Mn2ϩ (plus 5 mM 2-ME) was determined. Escherichia coli K-12 9784233 1.1.1.103 C38D 24937 25120 Nondenaturing PAGE of wild-type threonine dehydrogenase and mutant C38D. The mobilities of wild-type TDH and mutant C38D were consistent with both being tetrameric proteins (149 kDa). Escherichia coli K-12 9784233 1.1.1.103 C38D 25121 25352 Their migration patterns (mutant C38D, lane 2; wild-type TDH, lane 3) were intermediate between that of the dimeric form of bovine serum albumin (134 kDa; lane 1, upper band) and protocatechuate dioxygenase (␣4␤4, 199 kDa; lane 4). Escherichia coli K-12 9784233 1.1.1.103 C38D 25709 25870 A large portion of mutant C38D was removed by the buffer wash since Mn2ϩ ions, which are used to enhance the binding of wild-type TDH to this resin (6), were not Escherichia coli K-12 9784233 1.1.1.103 C38D 25967 26105 The elution profile of mutant C38D after applying a pulse of NADϩ to the affinity column was found to be similar to that of wild-type TDH. Escherichia coli K-12 9784233 1.1.1.103 C38D 26555 26650 The CD spectra of wild-type TDH and mutants C38S and C38D were virtually identical (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38S 26555 26650 The CD spectra of wild-type TDH and mutants C38S and C38D were virtually identical (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38D 26972 27193 The relative migration of mutant C38D (lane 2) and wild-type TDH (lane 3) was between that of the dimeric form of BSA (Mr 134,000; lane 1, upper band) and the ␣4␤4 form of protocatechuate dioxygenase (Mr 199,000; lane 4). Escherichia coli K-12 9784233 1.1.1.103 C38D 27965 28132 When subjected to size-exclusion chromatography, both mutants C38S and C38D were found to elute with the same retention volume as tetrameric wild-type TDH (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38S 27965 28132 When subjected to size-exclusion chromatography, both mutants C38S and C38D were found to elute with the same retention volume as tetrameric wild-type TDH (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38S 27965 28132 When subjected to size-exclusion chromatography, both mutants C38S and C38D were found to elute with the same retention volume as tetrameric wild-type TDH (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38D 27965 28132 When subjected to size-exclusion chromatography, both mutants C38S and C38D were found to elute with the same retention volume as tetrameric wild-type TDH (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38D 28313 28448 Whereas added Zn2ϩ has no influence on wild-type TDH, 5 mM Zn2ϩ increased the activity of mutant C38D almost 60-fold to 11.4 units/ mg. Escherichia coli K-12 9784233 1.1.1.103 C38D 28313 28448 Whereas added Zn2ϩ has no influence on wild-type TDH, 5 mM Zn2ϩ increased the activity of mutant C38D almost 60-fold to 11.4 units/ mg. Escherichia coli K-12 9784233 1.1.1.103 C38D 28449 28658 Unlike the activating effect it has on wild-type TDH activity (6), Mn2ϩ (1 mM) lowered the activity of mutant C38D by 50%; when included with 5 mM Zn2ϩ, 1 mM Mn2ϩ lessened the Zn2ϩ-stimulated activity by ϳ80%. Escherichia coli K-12 9784233 1.1.1.103 C38D 28449 28658 Unlike the activating effect it has on wild-type TDH activity (6), Mn2ϩ (1 mM) lowered the activity of mutant C38D by 50%; when included with 5 mM Zn2ϩ, 1 mM Mn2ϩ lessened the Zn2ϩ-stimulated activity by ϳ80%. Escherichia coli K-12 9784233 1.1.1.103 C38D 28807 28945 Co2ϩ (5 mM) increased activity 6-fold, whereas Hg2ϩ (0.5 mM) completely blocked catalysis of mutant C38D as it also does of wild-type TDH. Escherichia coli K-12 9784233 1.1.1.103 C38D 30362 30433 Another preparation of this mutant gave similar results to those shown. Escherichia coli K-12 9784233 1.1.1.103 C38S 31246 31515 Although no activity could be detected by usual assay procedures with mutant C38S, when this protein was preincubated with 1 mM Zn2ϩ and then assayed in a reaction mixture containing 1 to 15 mM Zn2ϩ, a low level of Zn2ϩ-dependent enzyme activity was measured (Fig. 3C). Escherichia coli K-12 9784233 1.1.1.103 C38D 31567 31817 pH/activity profile of native and Zn2ϩ-treated wildtype TDH as well as mutant C38D. Whereas purified mutant C38D shows quite low catalytic activity and a pH optimum near 7.5, it has optimal activity at pH 8.4 when stimulated by 5 mM Zn2ϩ (not shown). Escherichia coli K-12 9784233 1.1.1.103 C38D 32559 32848 When the reaction velocity of Zn2ϩ-activated mutant C38D was measured with 20 mM threonine and varying concentrations of NADϩ, an apparent Km value for NADϩ of 0.75 mM was determined (not shown), whose value is ninefold higher than the true Km for NADϩ reported for wild-type TDH (21, 25). Escherichia coli K-12 9784233 1.1.1.103 C38D 32559 32848 When the reaction velocity of Zn2ϩ-activated mutant C38D was measured with 20 mM threonine and varying concentrations of NADϩ, an apparent Km value for NADϩ of 0.75 mM was determined (not shown), whose value is ninefold higher than the true Km for NADϩ reported for wild-type TDH (21, 25). Escherichia coli K-12 9784233 1.1.1.103 C38D 32559 32848 When the reaction velocity of Zn2ϩ-activated mutant C38D was measured with 20 mM threonine and varying concentrations of NADϩ, an apparent Km value for NADϩ of 0.75 mM was determined (not shown), whose value is ninefold higher than the true Km for NADϩ reported for wild-type TDH (21, 25). Escherichia coli K-12 9784233 1.1.1.103 C38D 32849 32942 Substrate specificity of wild-type TDH and mutant C38D with or without activating metal ions. Escherichia coli K-12 9784233 1.1.1.103 C38D 35185 35395 Activity assays with mutant C38D contained 7.5 mM NADϩ and 20 mM of the L-isomer of each substrate (D-threonine was tested at 20 mM); similar results were obtained with two separate preparations of this mutant. Escherichia coli K-12 9784233 1.1.1.103 C38D 35532 35713 The levels of activity shown by mutant C38D with a single concentration of various substrates with no metal ion added or after activation with 5 mM Zn2ϩ are also listed in Table IV. Escherichia coli K-12 9784233 1.1.1.103 C38D 35878 36051 Whereas Zn2ϩstimulated mutant C38D showed a 40-fold increase in the rate of turnover of L-threonine, its activity with D,L-2-amino-3-hydroxypentanoate increased only 2-fold. Escherichia coli K-12 9784233 1.1.1.103 C38D 35878 36051 Whereas Zn2ϩstimulated mutant C38D showed a 40-fold increase in the rate of turnover of L-threonine, its activity with D,L-2-amino-3-hydroxypentanoate increased only 2-fold. Escherichia coli K-12 9784233 1.1.1.103 C38D 36052 36210 In general, Zn2ϩ-activated mutant C38D, like Mn2ϩ- or Cd2ϩ-activated wild-type TDH, is more active with threonine and less active with alternative substrates. Escherichia coli K-12 9784233 1.1.1.103 C38D 36052 36210 In general, Zn2ϩ-activated mutant C38D, like Mn2ϩ- or Cd2ϩ-activated wild-type TDH, is more active with threonine and less active with alternative substrates. Escherichia coli K-12 9784233 1.1.1.103 C38D 36052 36210 In general, Zn2ϩ-activated mutant C38D, like Mn2ϩ- or Cd2ϩ-activated wild-type TDH, is more active with threonine and less active with alternative substrates. Escherichia coli K-12 9784233 1.1.1.103 C38D 36052 36210 In general, Zn2ϩ-activated mutant C38D, like Mn2ϩ- or Cd2ϩ-activated wild-type TDH, is more active with threonine and less active with alternative substrates. Escherichia coli K-12 9784233 1.1.1.103 C38S 38843 39067 That mutants C38S and C38D could be purified to homogeneity from cell-free extracts of E. coli by the same procedure used to isolate wild-type TDH suggests that no gross conformational changes occurred in the mutant enzymes. Escherichia coli K-12 9784233 1.1.1.103 C38D 38843 39067 That mutants C38S and C38D could be purified to homogeneity from cell-free extracts of E. coli by the same procedure used to isolate wild-type TDH suggests that no gross conformational changes occurred in the mutant enzymes. Escherichia coli K-12 9784233 1.1.1.103 C38S 38843 39067 That mutants C38S and C38D could be purified to homogeneity from cell-free extracts of E. coli by the same procedure used to isolate wild-type TDH suggests that no gross conformational changes occurred in the mutant enzymes. Escherichia coli K-12 9784233 1.1.1.103 C38D 38843 39067 That mutants C38S and C38D could be purified to homogeneity from cell-free extracts of E. coli by the same procedure used to isolate wild-type TDH suggests that no gross conformational changes occurred in the mutant enzymes. Escherichia coli K-12 9784233 1.1.1.103 C38D 39068 39238 The CD spectra of these two mutants were virtually identical to that of wild-type TDH and, as isolated, they each contained one Zn2ϩ atom per subunit, like wild-type TDH. Escherichia coli K-12 9784233 1.1.1.103 C38S 39239 39531 Wild-type TDH— known to be a homotetrameric protein by several different methods (25, 27)—and mutant C38D showed identical electrophoretic mobilities when subjected to nondenaturing PAGE, and mutants C38S and C38D had the same elution volume as wild-type TDH in size-exclusion chromatography. Escherichia coli K-12 9784233 1.1.1.103 C38D 39239 39531 Wild-type TDH— known to be a homotetrameric protein by several different methods (25, 27)—and mutant C38D showed identical electrophoretic mobilities when subjected to nondenaturing PAGE, and mutants C38S and C38D had the same elution volume as wild-type TDH in size-exclusion chromatography. Escherichia coli K-12 9784233 1.1.1.103 C38D 39239 39531 Wild-type TDH— known to be a homotetrameric protein by several different methods (25, 27)—and mutant C38D showed identical electrophoretic mobilities when subjected to nondenaturing PAGE, and mutants C38S and C38D had the same elution volume as wild-type TDH in size-exclusion chromatography. Escherichia coli K-12 9784233 1.1.1.103 C38D 39239 39531 Wild-type TDH— known to be a homotetrameric protein by several different methods (25, 27)—and mutant C38D showed identical electrophoretic mobilities when subjected to nondenaturing PAGE, and mutants C38S and C38D had the same elution volume as wild-type TDH in size-exclusion chromatography. Escherichia coli K-12 9784233 1.1.1.103 C38E 39677 39978 Although mutant C38E could not be purified by the usual fractionation procedures, one notable result was obtained with an approximately 50% purified sample of this mutant; Cd2ϩ (but not Zn2ϩ) ions stimulated its nearly nondetectable level of activity in a saturable manner (activation Kd ϳ 4 ␮M Cd2ϩ). Escherichia coli K-12 9784233 1.1.1.103 C38S 39979 40239 Further work must still be done to better purify and characterize mutant C38E. Since EXAFS spectroscopy indicated that the one Zn2ϩ atom in wild-type TDH is at a structural 4-Cys site (4), the finding that mutant C38S was catalytically inactive was unexpected. Escherichia coli K-12 9784233 1.1.1.103 C38S 40240 40555 The observation, however, that mutant C38S catalyzes a low level of activity when high concentrations of Zn2ϩ are added to the usual assay mixture was the first indication that Cys-38 of E. coli TDH might serve as a ligand in a second, possibly “catalytic” metal-ion binding site comparable to Cys-46 in horse LADH. Escherichia coli K-12 9784233 1.1.1.103 C38S 40240 40555 The observation, however, that mutant C38S catalyzes a low level of activity when high concentrations of Zn2ϩ are added to the usual assay mixture was the first indication that Cys-38 of E. coli TDH might serve as a ligand in a second, possibly “catalytic” metal-ion binding site comparable to Cys-46 in horse LADH. Escherichia coli K-12 9784233 1.1.1.103 C38D 41454 41562 The active-site structure of Zn2ϩ-stimulated mutant C38D, therefore, does not seem to be adversely affected. Escherichia coli K-12 9784233 1.1.1.103 C38D 41563 41713 Of the divalent metal ions tested with mutant C38D, Zn2ϩ was the most stimulatory; Co2ϩ and Cd2ϩ increased catalytic activity to a much lesser extent. Escherichia coli K-12 9784233 1.1.1.103 C38D 41840 42089 Whereas Mn2ϩ stimulates the enzymatic activity of wild-type TDH, this metal ion decreased that of mutant C38D. Mutant C38D can apparently bind Mn2ϩ at or near the activating Zn2ϩ site since its Zn2ϩ-stimulated activity is lessened 90% by added Mn2ϩ. Escherichia coli K-12 9784233 1.1.1.103 C38D 41840 42089 Whereas Mn2ϩ stimulates the enzymatic activity of wild-type TDH, this metal ion decreased that of mutant C38D. Mutant C38D can apparently bind Mn2ϩ at or near the activating Zn2ϩ site since its Zn2ϩ-stimulated activity is lessened 90% by added Mn2ϩ. Escherichia coli K-12 9784233 1.1.1.103 C38D 42235 42401 Both Cd2ϩ- and Mn2ϩ-activated wild-type TDH and Zn2ϩ-stimulated mutant C38D are more active with the natural substrate, L-threonine, than with alternative substrates. Escherichia coli K-12 9784233 1.1.1.103 C38D 42235 42401 Both Cd2ϩ- and Mn2ϩ-activated wild-type TDH and Zn2ϩ-stimulated mutant C38D are more active with the natural substrate, L-threonine, than with alternative substrates. Escherichia coli K-12 9784233