Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase.
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.
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan.
Yu
Y
Yoshikane
Nana
N
Yokochi
Kouhei
K
Ohnishi
Hideyuki
H
Hayashi
Toshiharu
T
Yagi
D020561:Alphaproteobacteria / Q000201:enzymology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification; D000637:Transaminases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D000637:Transaminases; C022494:pyridoxamine-pyruvate transaminase