Inactivation of Mycobacterium tuberculosis L,D-transpeptidase Ldt Mt1 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 LdtMt1 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 LdtMt1, which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited LdtMt1, 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 LdtMt1, 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. Copyright © 2012, American Society for Microbiology. All Rights Reserved.