Characterization of D-amino acid aminotransferase from Lactobacillus salivarius

Abstract 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. salivarius d -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 30 min 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 K m values for d -alanine and α-ketoglutarate were 1.05 and 3.78 mM, 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. salivarius d -AAT thus differs greatly from those of the other d -AATs so far reported.