Molecular weight and subunit structure of pyridoxamine pyruvate transaminase

Kolb, Helmut J.; Cole, R D; Snell, Esmond E.

doi:10.1021/bi00848a035

Cited by 23 publications

(7 citation statements)

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“…Thus PN and PM, and some of their metabolites, may directly or indirectly induce the expression of all of the genes that form the gene cluster for the degradation of PN. The partially purified enzyme preparation gave a main protein band corresponding to a molecular mass of 41 kDa, which is similar to that of the Pseudomonas enzyme (38 kDa) [7], and several minor bands on an SDS/PAGE gel (Figure 1). The N-terminal amino acid sequence of the main protein was Met-Arg-Tyr-Pro-Xaa-Xaa-Ala.…”

Section: Other Analytical Methodsmentioning

confidence: 62%

“…The enzyme fraction eluted with the buffer containing 2 M KCl was used as the partially purified enzyme preparation. The partially purified enzyme was subjected to SDS/PAGE (10 % gel), and the protein component on the SDS/polyacrylamide gel exhibiting a subunit molecular mass corresponding to that of Pseudomonas PPAT [7] was transferred on to a PVDF membrane using a Trans-blot cell system (Bio-Rad). The transferred enzyme was subjected to N-terminal amino acid sequencing with an Applied Biosystems 492 protein sequencer.…”

Section: Partial Purification and N-terminal Amino Acid Sequencing Ofmentioning

confidence: 99%

“…PPAT (pyridoxamine-pyruvate aminotransferase) (EC 2.6.1.30), which is involved in a degradation pathway for PN (pyridoxine) [1], one of six natural vitamin B 6 compounds, is a PLP (pyridoxal 5 -phosphate)-independent aminotransferase that catalyses the transfer of an amino group between PM (pyridoxamine) and pyruvate in the forward reaction, and between PL (pyridoxal) and L-alanine in the reverse reaction. Because this reaction serves as a model for a half-reaction of the overall transamination reaction catalysed by general PLP-dependent aminotransferases, many studies have been performed on its enzymatic [2,3], kinetic [4][5][6] and molecular [7,8] properties. However, neither its primary structure nor the ppat gene encoding the enzyme has been reported.…”

Section: Introductionmentioning

confidence: 99%

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Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

Yoshikane

Yokochi

Ohnishi

et al. 2006

Biochemical Journal

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Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.

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Section: Other Analytical Methodsmentioning

confidence: 62%

Section: Partial Purification and N-terminal Amino Acid Sequencing Ofmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

Yoshikane

Yokochi

Ohnishi

et al. 2006

Biochemical Journal

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“…The partial amino acid sequence around the pyridoxalbinding site of this enzyme was different from that of the corresponding peptides that found from other pyridoxal phosphate-dependent enzymes. [4][5][6][7] Although, purification, characterization, and partial amino acid sequence determination of the PM-pyruvate transaminase of this bacteria has been reported, the nucleotide sequence of the gene encoding the enzyme and the protein structure are still unknown. Pyridoxine 4-oxidase, which catalyzes the reaction of PN to PL requires FAD as a coenzyme.…”

Section: Introductionmentioning

confidence: 99%

Untitled

Trongpanich¹,

Niamsanit²,

Sineenat³

2005

ScienceAsia

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Two different pathways, pathway I and II, for vitamin B-6 degradation are known in bacteria. Pyridoxamine-pyruvate transaminase and pyridoxine 4-oxidase, respectively, use pyridoxamine and pyridoxine as a substrate in pathway I of vitamin B-6 degradation. Among 500 bacterial isolates, four isolates, T2, T4, T5, and T6, were identified as the fast growing bacterial isolates using pyridoxine as a carbon source. The activities of pyridoxine 4-oxidase and pyridoxamine-pyruvate transaminase were determined by analyzing the reaction product, pyridoxal, by isocratic reverse-phase HPLC. Only two isolates, T2 and T6, showed pyridoxine 4-oxidase activities of 4.08 and 9.38 Unit/mg (pmol/min/mg protein), respectively. The activity of pyridoxamine-pyruvate transaminase was 6.31 Unit/mg in T2 isolate and 6.11 Unit/mg in T6 isolate. Some biochemical characteristics and nucleotide sequences (500 bases) of 16S rDNA suggested that T2 and T6 were closely related to Ochrobactrum anthropi and Enterobacter cloacae, respectively. This is the first report of pyridoxamine-pyruvate transaminase and pyridoxine 4-oxidase, enzymes in vitamin B-6 degradation pathway in O. anthropi and E. cloacae-like bacteria.

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“…and characterized (2). Many biochemical studies have been performed on it (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), because its reaction serves as a model for a half-reaction in the overall transamination reaction catalyzed by general PLP-dependent aminotransferases. Recently, we first identified the gene encoding PPAT in a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti MAFF303099, characterized with the recombinant PPAT overexpressed in Escherichia coli cells (13), and found that it belonged to the class V aminotransferases of fold type I of PLPdependent enzymes (14), although it does not use PLP as a coenzyme.…”

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confidence: 99%

Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099

Yoshikane

Yokochi

Yamasaki

et al. 2008

Journal of Biological Chemistry

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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 Å 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 Å resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven ␤-sheets and eight ␣-helices, and a smaller C-terminal domain, consisting of three ␤-sheets and four ␣-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The ␣-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.Pyridoxamine-pyruvate aminotransferase (PPAT, EC 2.6.1.30) 2 is a pyridoxal 5Ј-phosphate (PLP)-independent aminotransferase that catalyzes the transfer of an amino group between pyridoxamine (PM) and pyruvate in the forward reaction and between pyridoxal (PL) and L-alanine in the reverse reaction. PPAT is involved in a degradation pathway for PM, one of six natural vitamin B 6 compounds (1). It has been purified from Pseudomonas sp. and characterized (2). Many biochemical studies have been performed on it (3-12), because its reaction serves as a model for a half-reaction in the overall transamination reaction catalyzed by general PLP-dependent aminotransferases. Recently, we first identified the gene encoding PPAT in a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti MAFF303099, characterized with the recombinant PPAT overexpressed in Escherichia coli cells (13), and found that it belonged to the class V aminotransferases of fold type I of PLPdependent enzymes (14), although it does not use PLP as a coenzyme.The recombinant PPAT bound the substrate PL at Lys-197 through a Schiff base, like general aminotransferases, forming an internal aldimine between PLP and an active site lysine residue, and the Schiff base formation was partially rate-determining in the reverse reaction. Sequence alignment of PPAT with other class V aminotransferases showed that the amino acid residues that have been shown to interact with PLP, based on the crystallography of E. coli phosphoserine aminotransferase (15) and human alanine-glyoxylate aminotransferase (AGAT; Ref. 16), were conserved in PPAT, and it was suggested that PL is held through interaction with these amino acid residues in the active site of PPAT (13). The predicted active...

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Molecular weight and subunit structure of pyridoxamine pyruvate transaminase

Cited by 23 publications

References 22 publications

Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

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Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099

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