The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform
Gerrit J Poelarends - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanisms and biocatalytic applications of Aspartate and methylAspartate Ammonia Lyases
ACS Chemical Biology, 2012Co-Authors: Marianne De Villiers, Vinod Puthan Veetil, Hans Raj, Jandre De Villiers, Gerrit J PoelarendsAbstract:Ammonia Lyases catalyze the formation of α,β-unsaturated bonds by the elimination of Ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes as catalysts for the synthesis of enantiomerically pure α-amino acids. In this Review Aspartate Ammonia Lyase and 3-methylAspartate Ammonia Lyase, which represent two different enzyme superfamilies, are discussed in detail. In the past few years, the three-dimensional structures of these Lyases in complex with their natural substrates have revealed the details of two elegant catalytic strategies. These strategies exploit similar deamination mechanisms that involve general-base catalyzed formation of an enzyme-stabilized enolate anion (aci-carboxylate) intermediate. Recent progress in the engineering and application of these enzymes to prepare enantiopure l-aspartic acid derivatives, which are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals and food additives, is also discussed.
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Structural basis for the catalytic mechanism of Aspartate Ammonia Lyase.
Biochemistry, 2011Co-Authors: Guntur Fibriansah, Vinod Puthan Veetil, Gerrit J Poelarends, Andy-mark W. H. ThunnissenAbstract:Aspartate Ammonia Lyases (or aspartases) catalyze the reversible deamination of L-Aspartate into fumarate and Ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-Aspartate at 2.4 and 2.6 A resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.
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site directed mutagenesis kinetic and inhibition studies of Aspartate Ammonia Lyase from bacillus sp ym55 1
FEBS Journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
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Site‐directed mutagenesis, kinetic and inhibition studies of Aspartate Ammonia Lyase from Bacillus sp. YM55‐1
The FEBS journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
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Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase
Chemistry (Weinheim an der Bergstrasse Germany), 2008Co-Authors: Barbara Weiner, Gerrit J Poelarends, Dick B. Janssen, Ben L. FeringaAbstract:The gene encoding Aspartate Ammonia Lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not affect AspB activity. The enzyme processes l-aspartic acid, but not d-aspartic acid, with a Km of ≈15 mM and a kcat of ≈40 s-1. By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The kcat values are comparable to those observed for the AspB-catalyzed addition of Ammonia to fumarate (≈90 s-1), whereas the Km values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97% ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
Jeewon Lee - One of the best experts on this subject based on the ideXlab platform.
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Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli.
The Biochemical journal, 2004Co-Authors: Yang-hoon Kim, Jin-seung Park, Jae-yong Cho, Kwang Myung Cho, Young-hoon Park, Jeewon LeeAbstract:The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of Aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (Aspartate Ammonia-Lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of Aspartate to fumarate and Ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-Lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, Aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
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Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli
Biochemical Journal, 2004Co-Authors: Yang-hoon Kim, Jin-seung Park, Jae-yong Cho, Kwang Myung Cho, Young-hoon Park, Jeewon LeeAbstract:The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of Aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (Aspartate Ammonia-Lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of Aspartate to fumarate and Ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine β-Lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, Aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
Dick B. Janssen - One of the best experts on this subject based on the ideXlab platform.
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site directed mutagenesis kinetic and inhibition studies of Aspartate Ammonia Lyase from bacillus sp ym55 1
FEBS Journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
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Site‐directed mutagenesis, kinetic and inhibition studies of Aspartate Ammonia Lyase from Bacillus sp. YM55‐1
The FEBS journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
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Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase
Chemistry (Weinheim an der Bergstrasse Germany), 2008Co-Authors: Barbara Weiner, Gerrit J Poelarends, Dick B. Janssen, Ben L. FeringaAbstract:The gene encoding Aspartate Ammonia Lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not affect AspB activity. The enzyme processes l-aspartic acid, but not d-aspartic acid, with a Km of ≈15 mM and a kcat of ≈40 s-1. By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The kcat values are comparable to those observed for the AspB-catalyzed addition of Ammonia to fumarate (≈90 s-1), whereas the Km values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97% ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
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biocatalytic enantioselective synthesis of n substituted aspartic acids by Aspartate Ammonia Lyase
Chemistry: A European Journal, 2008Co-Authors: Barbara Weiner, Gerrit J Poelarends, Dick B. Janssen, Ben L. FeringaAbstract:The gene encoding Aspartate Ammonia Lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His(6) tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His(6) tag does not affect AspB activity. The enzyme processes L-aspartic acid, but not D-aspartic acid, with a K(m) of approximately 15 mM and a k(cat) of approximately 40 s(-1). By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The k(cat) values are comparable to those observed for the AspB-catalyzed addition of Ammonia to fumarate ( approximately 90 s(-1)), whereas the K(m) values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
Vinod Puthan Veetil - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanisms and biocatalytic applications of Aspartate and methylAspartate Ammonia Lyases
ACS Chemical Biology, 2012Co-Authors: Marianne De Villiers, Vinod Puthan Veetil, Hans Raj, Jandre De Villiers, Gerrit J PoelarendsAbstract:Ammonia Lyases catalyze the formation of α,β-unsaturated bonds by the elimination of Ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes as catalysts for the synthesis of enantiomerically pure α-amino acids. In this Review Aspartate Ammonia Lyase and 3-methylAspartate Ammonia Lyase, which represent two different enzyme superfamilies, are discussed in detail. In the past few years, the three-dimensional structures of these Lyases in complex with their natural substrates have revealed the details of two elegant catalytic strategies. These strategies exploit similar deamination mechanisms that involve general-base catalyzed formation of an enzyme-stabilized enolate anion (aci-carboxylate) intermediate. Recent progress in the engineering and application of these enzymes to prepare enantiopure l-aspartic acid derivatives, which are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals and food additives, is also discussed.
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Structural basis for the catalytic mechanism of Aspartate Ammonia Lyase.
Biochemistry, 2011Co-Authors: Guntur Fibriansah, Vinod Puthan Veetil, Gerrit J Poelarends, Andy-mark W. H. ThunnissenAbstract:Aspartate Ammonia Lyases (or aspartases) catalyze the reversible deamination of L-Aspartate into fumarate and Ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-Aspartate at 2.4 and 2.6 A resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.
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site directed mutagenesis kinetic and inhibition studies of Aspartate Ammonia Lyase from bacillus sp ym55 1
FEBS Journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
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Site‐directed mutagenesis, kinetic and inhibition studies of Aspartate Ammonia Lyase from Bacillus sp. YM55‐1
The FEBS journal, 2009Co-Authors: Vinod Puthan Veetil, Hans Raj, Dick B. Janssen, Wim J. Quax, Gerrit J PoelarendsAbstract:Aspartate Ammonia Lyases (also referred to as aspartases) catalyze the reversible deamination of l-Aspartate to yield fumarate and Ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-Aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates Ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which l-Aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l-Aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for l-Aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the Lyases in the aspartase/fumarase superfamily.
Yang-hoon Kim - One of the best experts on this subject based on the ideXlab platform.
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Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli.
The Biochemical journal, 2004Co-Authors: Yang-hoon Kim, Jin-seung Park, Jae-yong Cho, Kwang Myung Cho, Young-hoon Park, Jeewon LeeAbstract:The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of Aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (Aspartate Ammonia-Lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of Aspartate to fumarate and Ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-Lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, Aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
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Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli
Biochemical Journal, 2004Co-Authors: Yang-hoon Kim, Jin-seung Park, Jae-yong Cho, Kwang Myung Cho, Young-hoon Park, Jeewon LeeAbstract:The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of Aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (Aspartate Ammonia-Lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of Aspartate to fumarate and Ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine β-Lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, Aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
