7XLL

Alanine racemase from Lactobacillus sakei Uonuma-1.

Summary for 7XLL

• Entry DOI: 10.2210/pdb7xll/pdb
• Descriptor: Alanine racemase, ACETATE ION, DI(HYDROXYETHYL)ETHER, ... (5 entities in total)
• Functional Keywords: alanine racemase, plp-dependent enzyme, isomerase
• Biological source: Latilactobacillus sakei
• Total number of polymer chains: 2
• Total formula weight: 84454.55

Authors

Shimizu-Ibuka, A.,Kato, Y. (deposition date: 2022-04-22, release date: 2023-03-01, Last modification date: 2023-11-29)

Primary citation

Shimizu-Ibuka, A.,Sato, A.,Ichimura, H.,Hiraga, H.,Nakayama, S.,Nishiwaki, T.
Regulation of alanine racemase activity by carboxylates and the d-type substrate d-alanine.
Febs J., 290:2954-2967, 2023

PubMed 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 , 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.

PubMed: 36732053
DOI: 10.1111/febs.16745

Experimental method

X-RAY DIFFRACTION (1.76 Å)