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 K m , 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 carboxylatebinding 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.

Effects of d-alanine Intake on Amino Acid Metabolism and Kidney Function in Healthy Adults: A Multicenter, Randomized Pilot Study

“…In particular, fermented foods are known to contain high amounts of d -amino acids, which are produced during microbial fermentation [ 22 ]. Furthermore, some lactic acid bacteria synthesize d -alanine and encode alanine racemase, which catalyzes the conversion of l -alanine to d -alanine [ 23 , 24 ]. Although we did not evaluate the d -alanine levels in stool, the protocol of the current study restricted the intake of fermented foods and the use of antimicrobial agents and lactic acid preparations during the follow-up period to minimize their influences.…”