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Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

Kobayashi, Jyumpei, Shimizu, Yasuhiro, Mutaguchi, Yuta, Doi, Katsumi, Ohshima, Toshihisa
Journal of Molecular Catalysis. B, Enzymatic 2013 v.94 pp. 15-22
Escherichia coli, Lactobacillus salivarius, alpha-ketoglutaric acid, catalytic activity, databases, gene expression, genes, lactic acid bacteria, pH, substrate specificity, temperature, transaminases
We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivariusd-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60°C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30min at 30°C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The Km values for d-alanine and α-ketoglutarate were 1.05 and 3.78mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivariusd-AAT thus differs greatly from those of the other d-AATs so far reported.