fThe Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by L,D-transpeptidases (LDTs), which are efficiently inactivated by a single ␤-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the ␤-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five L,D-transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), ␤-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the crosslinking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the ␤-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.

␤-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.

Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of ␤-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 ␤-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new ␤-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 M ؊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.␤-Lactam antibiotics entered clinical trials in 1941 and have become and remained the most widely used family of drugs for the treatment of severe infections. The success of these molecules as therapeutic agents originates from a combination of properties, including low toxicity, excellent bioavailability, and broad-spectrum bactericidal activity. The latter property is accounted for by the conservation of the target, the active-site serine DD-transpeptidases, thought to catalyze an essential step in cell wall synthesis in all peptidoglycan-containing bacteria (1). The discovery of hundreds of ␤-lactams and of ␤-lactamase inhibitors has made it possible to partially compensate for the erosion of antibacterial activity due to the emergence of various mechanisms of resistance. In Gram-negative bacteria, these mechanisms mostly involve the production of ␤-lactamases, often associated with decreased outer membrane permeability and drug efflux. In Gram-positive bacteria, ␤-lactamase production is also frequent, but modification of the DD-transpeptidases is the clinically relevant mechanism in important pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae, and the enterococci. More recently, bypass of the DD-transpeptidases by a novel class of peptidoglycan polymerases, the LDtranspeptidases, has been shown to convey high level resistance to all ␤-lactams, except the carbapenems, in mutants of Enterococcus faecium selected in vitro (2). Transpeptidases of the DD and LD specificities are structurally unrelated, contain different active-site nucleophiles (Ser versus Cys, respectively), and catalyze formation of different peptidoglycan cross-links (433 versus 333, respectively) (3). The two...

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a biopolymer highly cross-linked through d,d–transpeptidation. Peptidoglycan cross-linking is catalyzed by Penicillin-Binding Proteins (PBPs) that are the essential target of β-lactam antibiotics. PBPs are functionally replaced by l,d–transpeptidases (Ldts) in ampicillin– resistant mutants of Enterococcus faecium and in wild-type Mycobacterium tuberculosis. Ldts are inhibited in vivo by a single class of β-lactams, the carbapenems, which act as a suicide substrate. We present here the first structure of a carbapenem–acylated l,d–transpeptidase, E. faecium Ldtfm acylated by ertapenem, which revealed key contacts between the carbapenem core and residues of the catalytic cavity of the enzyme. Significant reorganization of the antibiotic conformation occurs upon enzyme acylation. These results, together with the analysis of protein–to–carbapenem proton transfers, provide new insight into the mechanism of Ldt acylation by carbapenems.

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an L,D-transpeptidase from Bacillussubtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains -the catalytic domain as well as the proposed peptidoglycan recognition domain -are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting L,D-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

Combinations of ␤-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum ␤-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of ␤-lactamase Bla MAb

Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.