Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

Abstract

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.

Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099

“…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 site structure of PPAT suggested that PLP cannot bind to the enzyme because the bulky side chain of an amino acid residue located in the phosphate-binding space in PLP-dependent aminotransferases prevents the approach of PLP to the active site of PPAT (13).…”

“…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 site structure of PPAT suggested that PLP cannot bind to the enzyme because the bulky side chain of an amino acid residue located in the phosphate-binding space in PLP-dependent aminotransferases prevents the approach of PLP to the active site of PPAT (13). One of the aims of this study was to determine the amino acid residue preventing the binding of PLP, and the extent to which PPAT can be converted to PMP-pyruvate (PLP-L-alanine) aminotransferase through mutation of a relevant residue.…”

“…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.…”

Gene Identification and Characterization of the Pyridoxine Degradative Enzyme .ALPHA.-(N-Acetylaminomethylene)succinic Acid Amidohydrolase from Mesorhizobium loti MAFF303099

“…The first enzyme for degradation of pyridoxine is pyridoxine 4-oxidase ( 4 , 5 ) and that for pyridoxamine is pyridoxamine-pyruvate aminotransferase ( 6 ). The second, third, seventh enzymes are pyridoxal 4-dehydrogenase ( 7 ), 4-pyridoxolactonase ( 8 ), and MHPC dioxygenase ( 9 ), respectively.…”

“…1B. The enzyme reaction is the final step in degradation pathway I for vitamin B 6 (pyridoxine, pyridoxamine, and pyridoxal) ( 1 ). The enzyme was purified from the crude extract of Pseudomonas MA-1 cells grown on pyridoxine as a sole carbon source, and some properties were characterized ( 2 , 3 ).…”