Conversion of the catalytic specificity of alanine racemase to a d-amino acid aminotransferase activity by a double active-site mutation

Alanine racemase depending on pyridoxal 5′-phosphate catalyzes the interconversion between d- and l-alanine. The enzyme from Bacillus stearothermophilus catalyzes the transamination as a side reaction with both substrates once per 3×107 times of the racemization. In this work, we studied the effects of the mutation of Arg219, and that of Arg219 and Tyr265 on the catalysis of Bacillus alanine racemase. Arg219 interacting with pyridinium nitrogen of the cofactor is conserved in all alanine racemases. The corresponding residue of aminotransferases is an acidic residue, such as glutamate or aspartate. Mutation of Arg219 to a glutamyl residue resulted in a 5.4-fold increase in the forward half transamination activity with d-alanine and a 103-fold decrease in the racemase activity. The double mutation, Arg219→Glu and Tyr265→Ala, completely abolished the racemase activity and increased the forward half transaminase activity 6.6-fold. Arg219 is one of the structural determinants of the catalytic specificity of the alanine racemase.