title: Inactivation of Mycobacterium tuberculosis l,d-transpeptidase LdtMt₁ by carbapenems and cephalosporins.
abstract: The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase Ldt(Mt1) by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt(Mt1), which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt(Mt1), with a binding rate constant of 0.08 μM(-1) min(-1). The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min(-1)) in comparison to the acylation rate constant (3.1 min(-1)). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10(-3) min(-1). Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt(Mt1) inactivator. Cephalosporins also formed covalent adducts with Ldt(Mt1), although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.
authors.affiliation: Centre de Recherche des Cordeliers, LRMA, Equipe 12, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.forename: Sébastien
authors.initials: S
authors.lastname: Triboulet
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.forename: Mélanie
authors.initials: M
authors.lastname: Ethève-Quelquejeu
authors.forename: Laurent
authors.initials: L
authors.lastname: Gutmann
authors.forename: Arul
authors.initials: A
authors.lastname: Marie
authors.forename: Lionel
authors.initials: L
authors.lastname: Dubost
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015780:Carbapenems / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D019818:Clavulanic Acid / Q000378:metabolism / Q000494:pharmacology; D054908:Extensively Drug-Resistant Tuberculosis / Q000188:drug therapy; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013845:Thienamycins / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D010457:Peptidoglycan; D013845:Thienamycins; D019818:Clavulanic Acid; D010458:Peptidyl Transferases; D000077731:Meropenem