Characterization of the extended substrate spectrum of the class A β-lactamase CESS-1 from Stenotrophomonas sp. and structure-based investigation into its substrate preference

Abstract

Objectives: Stenotrophomonas spp. intrinsically resistant to many /3-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A /3-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1. Methods: The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography. Results: CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were 1249.6 s-1 , 204.3 s-1 , and 69.8 s-1 , respectively, with the accompanying KM values of 287.6 mu M, 236.7 mu M, and 28.8 mu M, respectively. Conclusions: CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the /35- /36 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar /3-lactam antibiotics. (c) 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.