title: Inactivation kinetics of a new target of beta-lactam antibiotics.
abstract: Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of β-lactam antibiotics. However, unrelated cysteine LD-transpeptidases have been recently recognized as a predominant mode of peptidoglycan cross-linking in Mycobacterium tuberculosis and as a bypass mechanism conferring resistance to all β-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new β-lactam target showed that acylation of the E. faecium enzyme (Ldt(fm)) by imipenem is irreversible. Using fluorescence kinetics, an original approach was developed to independently determine the catalytic constants for imipenem binding (k(1) = 0.061 μM(-1) min(-1)) and acylation (k(inact) = 4.5 min(-1)). The binding step was limiting at the minimal drug concentration required for bacterial growth inhibition. The Michaelis complex was committed to acylation because its dissociation was negligible. The emergence of imipenem resistance involved substitutions in Ldt(fm) that reduced the rate of formation of the non-covalent complex but only marginally affected the efficiency of the acylation step. The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis.
authors.affiliation: Centre de Recherche des Cordeliers, Laboratoire de Recherche Moléculaire sur les Antibiotiques, Equipe 12, Université Pierre et Marie Curie, Paris 6, UMR S 872, Paris, F-75006 France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Triboulet
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.forename: Carole
authors.initials: C
authors.lastname: Veckerlé
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.forename: Angèle
authors.initials: A
authors.lastname: Nguekam-Moumi
authors.forename: Laurent
authors.initials: L
authors.lastname: Gutmann
authors.forename: Louis B
authors.initials: LB
authors.lastname: Rice
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D016984:Enterococcus faecium / Q000201:enzymology*; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D047090:beta-Lactams / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; D000217:Acyltransferases