␤-Lactams are usually not considered for treatment of tuberculosis (TB) since Mycobacterium tuberculosis produces a species-specific broad-spectrum class A ␤-lactamase (BlaC) (12). In spite of BlaC, carbapenems and the combination of amoxicillin and clavulanic acid have been reported to be bactericidal in vitro (4,5,7) and to reduce the burden of M. tuberculosis in the sputum of patients with pulmonary tuberculosis (4, 5). However, clinical assessments of the drugs for treatment of multidrug-resistant tuberculosis (MDR-TB) are limited to anecdotal cases involving combined therapy with second-line drugs (23, 24). The potential interest in ␤-lactams for the treatment of extensively drug-resistant tuberculosis (XDR-TB) has recently been renewed by detailed characterization of BlaC that showed that this ␤-lactamase is irreversibly inactivated by clavulanic acid and hydrolyzes carbapenems only at a low rate (12, 13). Combined with clavulanic acid, carbapenems are not only bactericidal against exponentially growing M. tuberculosis but are also active against nonreplicating forms of the bacilli (13). Furthermore, the combination was uniformly active against a collection of XDR strains (13).The main target of meropenem in M. tuberculosis is unlikely to be the D,D-transpeptidase activity of classical penicillin-binding proteins (PBPs) since the peptidoglycan of this bacterium contains a high proportion (80%) of cross-links connecting residues at the third position of stem peptides (3¡3 cross-links) (Fig. 1A) (15). These cross-links are formed by L,D-transpeptidases (Ldts) and replace the 4¡3 cross-links synthesized by PBPs (Fig. 1B). Ldts and PBPs are structurally unrelated and contain active-site cysteine and serine residues, respectively (2, 19). The chromosome of M. tuberculosis strain H37Rv encodes five L,D-transpeptidases (Ldts) designated Ldt Mt1 to Ldt Mt5 . Among these paralogues, Ldt Mt1 and Ldt Mt2 are both functional in an in vitro peptidoglycan cross-linking assay and inactivated by carbapenems (10, 15). Ldt Mt2 is essential for virulence in a mouse model of acute infection (10), whereas Ldt Mt1 is thought to play a critical role in peptidoglycan adaptation to the nonreplicative state of the bacilli (15).Mass spectrometry analyses have previously shown that carbapenems bind covalently to Ldt Mt1 and Ldt Mt2 (Fig. 1C) (10, 15), but the kinetics of this reaction have not been explored and the interaction of L,D-transpeptidases with drugs belonging to other ␤-lactam classes has not been investigated in detail. Here, we investigate the mechanism and kinetics of Ldt Mt1 inactivation by four carbapenems (meropenem, doripenem, imipenem, and ertapenem) and three cephalosporins (cefotaxime, cephalothin, and ceftriaxone) using a combination of mass spectrometry and stopped-flow fluorescence spectroscopy. We show that both classes of drugs form covalent adducts with Ldt Mt1 , although enzyme acylation with cephalosporins is slower and leads to the

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldtfm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldtfm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldtfm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldtfm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldtfm acylation was followed by rupture of the C5–C6 bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldtfm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.