Role of the Arginine 214 in the substrate specificity of OXA-48.

Role of the Arginine 214 in the substrate specificity of OXA-48. Antimicrob Agents Chemother. 2020 Feb 18;: Authors: Oueslati S, Retailleau P, Marchini L, Berthault C, Dortet L, Bonnin RA, Iorga BI, Naas T Abstract Increasing number of variants of the carbapenem-hydrolyzing class D β-lactamase OXA-48 are identified in Enterobacterales worldwide. Among them, OXA-181 and OXA-232 are of particular interest, as they differ from each other by a single amino-acid (AA) substitution at position 214 (R in OXA-181, and S in OXA-232), that results in reduced carbapenem-hydrolyzing activity for OXA-232. To investigate the role of the AA214, the X-ray structure of OXA-232 was determined and the AA214 of OXA-48 and of OXA-232 was replaced by G, L, D, E, S, R, and K using site-directed mutagenesis. These mutants were phenotypically characterized, and three mutants of OXA-232 were purified to study their steady-state kinetic properties. X-ray structure of OXA-232 along with molecular modelling studies showed that the interaction via a salt bridge between R214 and D159 in OXA-48 is not possible with G214 or S214 mutations. In contrast, with K214 that is also positively charged, the interaction with D159 is maintained. With the E214 mutant an alternative binding conformation of imipenem was evidenced that is not compatible with a nucleophilic attack by S70. Thus, imipenem has very poor apparent affinity for the E214 mutant because of its non-producti...
Source: Antimicrobial Agents and Chemotherapy - Category: Microbiology Authors: Tags: Antimicrob Agents Chemother Source Type: research