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Toshiharu Yagi - One of the best experts on this subject based on the ideXlab platform.
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engineering mesorhizobium loti pyridoxamine pyruvate aminotransferase for production of pyridoxamine with l glutamate as an amino donor
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Yu Yoshikane, Nana Yokochi, Asuka Tamura, Khalil Ellouze, Eitora Yamamura, Hanae Mizunaga, Noboru Fujimoto, Keiji Sakamoto, Yoshihiro Sawa, Toshiharu YagiAbstract:Abstract Pyridoxamine–pyruvate aminotransferase (PPAT), a novel Pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between pyridoxamine and pyruvate to generate Pyridoxal and l -alanine. The enzyme can be used for synthesis of pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l -alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l -glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l -glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl- l -glutamate showed that γ-carboxyl group of l -glutamate was hydrogen-bound with an imidazole group of His35. The production of pyridoxamine from Pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of Pyridoxal into pyridoxamine at 30 °C with l -glutamate as an amino donor.
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crystal structure of pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5′-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5'-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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An enzymatic fluorometric assay for Pyridoxal with high specificity and sensitivity.
Bioscience biotechnology and biochemistry, 2002Co-Authors: Yanee Trongpanich, Mitsunori Mito, Toshiharu YagiAbstract:An enzymatic fluorometric assay for Pyridoxal with Pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of Pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for Pyridoxal, and thus animal samples could be directly analyzed without separation of Pyridoxal 5'-phosphate by column chromatography.
Yu Yoshikane - One of the best experts on this subject based on the ideXlab platform.
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engineering mesorhizobium loti pyridoxamine pyruvate aminotransferase for production of pyridoxamine with l glutamate as an amino donor
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Yu Yoshikane, Nana Yokochi, Asuka Tamura, Khalil Ellouze, Eitora Yamamura, Hanae Mizunaga, Noboru Fujimoto, Keiji Sakamoto, Yoshihiro Sawa, Toshiharu YagiAbstract:Abstract Pyridoxamine–pyruvate aminotransferase (PPAT), a novel Pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between pyridoxamine and pyruvate to generate Pyridoxal and l -alanine. The enzyme can be used for synthesis of pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l -alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l -glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l -glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl- l -glutamate showed that γ-carboxyl group of l -glutamate was hydrogen-bound with an imidazole group of His35. The production of pyridoxamine from Pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of Pyridoxal into pyridoxamine at 30 °C with l -glutamate as an amino donor.
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crystal structure of pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5′-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5'-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
Nana Yokochi - One of the best experts on this subject based on the ideXlab platform.
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engineering mesorhizobium loti pyridoxamine pyruvate aminotransferase for production of pyridoxamine with l glutamate as an amino donor
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Yu Yoshikane, Nana Yokochi, Asuka Tamura, Khalil Ellouze, Eitora Yamamura, Hanae Mizunaga, Noboru Fujimoto, Keiji Sakamoto, Yoshihiro Sawa, Toshiharu YagiAbstract:Abstract Pyridoxamine–pyruvate aminotransferase (PPAT), a novel Pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between pyridoxamine and pyruvate to generate Pyridoxal and l -alanine. The enzyme can be used for synthesis of pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l -alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l -glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l -glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl- l -glutamate showed that γ-carboxyl group of l -glutamate was hydrogen-bound with an imidazole group of His35. The production of pyridoxamine from Pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of Pyridoxal into pyridoxamine at 30 °C with l -glutamate as an amino donor.
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crystal structure of pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5′-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5'-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
Hideyuki Hayashi - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5′-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5'-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
Bunzo Mikami - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of pyridoxamine pyruvate aminotransferase from mesorhizobium loti maff303099
Journal of Biological Chemistry, 2008Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Abstract Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5′-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
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Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099
The Journal of biological chemistry, 2007Co-Authors: Yu Yoshikane, Kimihiko Mizutani, Bunzo Mikami, Nana Yokochi, Kouhei Ohnishi, Masayuki Yamasaki, Hideyuki Hayashi, Toshiharu YagiAbstract:Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a Pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form Pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, Pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate Pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of Pyridoxal 5'-phosphate and made PPAT specific to Pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.
