Regulation of alanine racemase activity by carboxylates and the d‐type substrate d‐alanine

Abstract

Alanine racemases (ALRs) are essential for d‐alanine (d‐Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short‐chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in Km, indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate‐binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate‐binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K‐mediated activation in the presence of d‐Ala at high concentrations, implying that d‐Ala also exhibits low‐affinity binding in the first carboxylate‐binding site and prevents carboxylate/G134K‐induced activation. Such regulation of enzyme activity by carboxylates and d‐Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.