Dynamics Induced by β-Lactam Antibiotics in the Active Site of Bacillus subtilis L,D-Transpeptidase

beta-lactams inhibit peptidoglycan polymerization by acting as suicide substrates of essential D,D-transpeptidases. Bypass of these enzymes by unrelated L,D-transpeptidases results in beta-lactam resistance, although carbapenems remain unexpectedly active. To gain insight into carbapenem specificity of L,D-transpeptidases (Ldts), we solved the nuclear magnetic resonance (NMR) structures of apo and imipenem-acylated Bacillus subtilis Ldt and show that the cysteine nucleophile is present as a neutral imidazole-sulfhydryl pair in the substrate-free enzyme. NMR relaxation dispersion does not reveal any preexisting conformational exchange in the apoenzyme, and change in flexibility is not observed upon noncovalent binding of beta-lactams (K-D > 37.5 mM). In contrast, covalent modification of active cysteine by both carbapenems and 2-nitro-5-thiobenzoate induces backbone flexibility that does not result from disruption of the imidazole-sulfhydryl proton interaction or steric hindrance. The chemical step of the reaction determines enzyme specificity since no differences in drug affinity were observed.