title: Dynamics induced by β-lactam antibiotics in the active site of Bacillus subtilis L,D-transpeptidase.
abstract: β-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 β-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 β-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.
authors.affiliation: CEA, DSV, Institut de Biologie Structurale (IBS), Grenoble F-38027, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Lecoq
authors.forename: Catherine
authors.initials: C
authors.lastname: Bougault
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.forename: Carole
authors.initials: C
authors.lastname: Veckerlé
authors.forename: Ombeline
authors.initials: O
authors.lastname: Pessey
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Simorre
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009579:Nitrobenzoates / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D047090:beta-Lactams / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D047090:beta-Lactams; C011136:thionitrobenzoic acid; D010458:Peptidyl Transferases; D003545:Cysteine