Non-classical transpeptidases yield insight into new antibacterials (original) (raw)
Related papers
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.
ACS Infectious Diseases
Commercial carbapenem antibiotics are being used to treat multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis. Like other β-lactams, carbapenems are irreversible inhibitors of serine D,D-transpeptidases involved in peptidoglycan biosynthesis. In addition to D,D-transpeptidases, mycobacteria also utilize nonhomologous cysteine L,D-transpeptidases (Ldts) to crosslink the stem peptides of peptidoglycan, and carbapenems form longlived acyl-enzymes with Ldts. Commercial carbapenems are C2 modifications of a common scaffold. This study describes the synthesis of a series of atypical, C5α modifications of the carbapenem scaffold, microbiological evaluation against Mycobacterium tuberculosis (Mtb) and the nontuberculous mycobacterial species, Mycobacterium abscessus (Mab), as well as acylation of an important mycobacterial target Ldt, Ldt Mt2. In vitro evaluation of these C5α-modified carbapenems revealed compounds with standalone (i.e., in the absence of a β-lactamase inhibitor) minimum inhibitory concentrations (MICs) superior to meropenem-clavulanate for Mtb, and meropenem-avibactam for Mab. Time-kill kinetics assays showed better killing (2−4 log decrease) of Mtb and Mab with lower concentrations of compound 10a as compared to meropenem. Although susceptibility of clinical isolates to meropenem varied by nearly 100-fold, 10a maintained excellent activity against all Mtb and Mab strains. High resolution mass spectrometry revealed that 10a acylates Ldt Mt2 at a rate comparable to meropenem, but subsequently undergoes an unprecedented carbapenem fragmentation, leading to an acyl-enzyme with mass of Δm = +86 Da. Rationale for the divergence of the nonhydrolytic fragmentation of the Ldt Mt2 acyl-enzymes is proposed. The observed activity illustrates the potential of novel atypical carbapenems as prospective candidates for treatment of Mtb and Mab infections.
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.
2014
The genome of Mycobacterium tuberculosis contains a gene, blaC, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme−drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent K m and k cat values of 0.8 μM and 0.03 min −1 , respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tightbinding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl−enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC−tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC−tebipenem Michaelis− Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem− BlaC structures.
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.
Antimicrobial agents and chemotherapy, 2017
As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by non-classical transpeptidases, namely the L,D-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of β-lactams determines their activity against M. tuberculosis Here, we studied the interactions of β-lactams with two L,D-transpeptidases in M. abscessus, namely LdtMab1 and LdtMab2, and found both the carbapenem and cephalosporin--but not penicillin-sub-class of β-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with β-lactams is redundant, doripenem and cefdinir exhibit synergy against both...
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,...