Chemical shift perturbations induced by the acylation of Enterococcus faecium l,d-transpeptidase catalytic cysteine with ertapenem (original) (raw)
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ACS Chemical Biology, 2013
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 βlactams, the carbapenems, which act as a suicide substrate. We present here the first structure of a carbapenem-acylated L,Dtranspeptidase, E. faecium Ldt fm 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 insights into the mechanism of Ldt acylation by carbapenems.
Biomolecular NMR assignments, 2012
The D: ,D: -transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving L: ,D: -transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the L: ,D: -transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the L: ,D: -transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the L: ,D: -transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain (1)H,...
Non-classical transpeptidases yield insight into new antibacterials
Nature chemical biology, 2017
Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the β-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of β-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for β-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activ...
BMC biochemistry, 2017
The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb). Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases. The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enz...
Structure, 2012
With multidrug-resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug targets are becoming an urgent need. Traditional b-lactam antibiotics that inhibit D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on L,Dtranspeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase Ldt Mt2 containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for Ldt Mt2 in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights to facilitate development of drugs targeting this validated yet unexploited enzyme.
Dynamics Induced by β-Lactam Antibiotics in the Active Site of Bacillus subtilis l,d-Transpeptidase
Structure, 2012
b-lactams inhibit peptidoglycan polymerization by acting as suicide substrates of essential D,D-transpeptidases. Bypass of these enzymes by unrelated L,D-transpeptidases results in b-lactam resistance, although carbapenems remain unexpectedly active. To gain insight into carbapenem specificity of L,D-transpeptidases (Ldts), we solved the nuclear magnetic resonance (NMR) structures of apo and imipenem-acylated Bacillus subtilis Ldt and show that the cysteine nucleophile is present as a neutral imidazole-sulfhydryl pair in the substrate-free enzyme. NMR relaxation dispersion does not reveal any preexisting conformational exchange in the apoenzyme, and change in flexibility is not observed upon noncovalent binding of b-lactams (K D > 37.5 mM). In contrast, covalent modification of active cysteine by both carbapenems and 2-nitro-5-thiobenzoate induces backbone flexibility that does not result from disruption of the imidazole-sulfhydryl proton interaction or steric hindrance. The chemical step of the reaction determines enzyme specificity since no differences in drug affinity were observed. Structure b-Lactams Induce Dynamics in L,D-Transpeptidases Structure 20, 850-861, May 9,
Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
PLoS ONE, 2013
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 (Ldt fm ) conveys high-level resistance to b-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin .2,000 mg/ml. Unexpectedly, Ldt fm does not confer resistance to b-lactams of the carbapenem class (imipenem MIC = 0.5 mg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 mg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify b-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt fm by formation of a thioester bond between the active-site cysteine and the b-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt fm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt fm acylation was followed by rupture of the C 5 -C 6 bond of the b-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 Ldt fm 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 b-lactam scaffold for L,D-transpeptidaseinactivation. Citation: Triboulet S, Dubée V, Lecoq L, Bougault C, Mainardi J-L, et al. (2013) Kinetic Features of L,D-Transpeptidase Inactivation Critical for b-Lactam Antibacterial Activity. PLoS ONE 8(7): e67831.
Atomic Model of a Cell-Wall Cross-Linking Enzyme in Complex with an Intact Bacterial Peptidoglycan
Journal of the American Chemical Society, 2014
The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly crosslinked 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.