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

Autor: Shimizu-Ibuka A; Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan.; Department of Biological Sciences, Faculty of Science, Kanagawa University, Japan., Sato A; Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan., Ichimura H; Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan., Hiraga H; Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan., Nakayama S; Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan., Nishiwaki T; Niigata Agricultural Research Institute Food Research Center, Japan.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2023 Jun; Vol. 290 (11), pp. 2954-2967. Date of Electronic Publication: 2023 Feb 15.
DOI: 10.1111/febs.16745
Abstrakt: 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 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.
(© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
Databáze: MEDLINE
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