Characterization of D-amino acid aminotransferase from Lactobacillus salivarius

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 alpha-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 degrees C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 degrees 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 alpha-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K-m values for D-alanine and alpha-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 alpha-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than alpha-ketoglutarate. The substrate specificity of L. salivarius D-AAT thus differs greatly from those of the other D-AATs so far reported. (c) 2013 Elsevier B.V. All rights reserved.