Targeting the Cell Wall of Mycobacterium tuberculosis: Structure and Mechanism of L,D-Transpeptidase 2

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Structure (Impact Factor: 5.62). 10/2012; 20(12). DOI: 10.1016/j.str.2012.09.016
Source: PubMed


With multidrug-resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug targets are becoming an urgent need. Traditional β-lactam antibiotics that inhibit D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on L,D-transpeptidases 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.

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    • "Since Mtb possess enzymes with L,D-and D,D-transpeptidase activities, both need to be inhibited simultaneously to comprehensively inhibit biosynthesis of the peptidoglycan layer and subsequently kill the bacteria. The L,D-transpeptidase has since then attracted the attention of several researchers (Böth et al., 2013; Cordillot et al., 2013; Correale, Ruggiero, Capparelli, Pedone, & Berisio, 2013; Correale, Ruggiero, Pedone, & Berisio, 2013; De & McIntosh, 2012; Dubee et al., 2012; Erdemli et al., 2012; Kim et al., 2013; Lecoq et al., 2012, 2013; Li et al., 2013; Sanders, Wright, & Pavelka, 2014; Schoonmaker et al., 2014; Triboulet et al., 2013). We have determined the experimental binding free energies of imipenem and meropenem (see Figure 1) to ex-Ldt Mtb2 utilizing isothermal titration calorimetry experiments (Erdemli et al., 2012). "
    Dataset: JBSD 3

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    • "Penicillin-binding proteins (Sauvage et al. 2008) and Ldts (Bielnicki et al. 2005; Biarrotte-Sorin et al. 2006; Erdemli et al. 2012) are structurally unrelated, with drastically different folds and catalytic sites (active nucleophiles are a serine in PBPs and a cysteine in Ldts). The two enzyme families also show different susceptibility towards b-lactam antibiotics. "
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    ABSTRACT: Penicillin-binding proteins were long considered as the only peptidoglycan cross-linking enzymes and one of the main targets of β-lactam antibiotics. A new class of transpeptidases, the L,D-transpeptidases, has emerged in the last decade. In most Gram-negative and Gram-positive bacteria, these enzymes generally have nonessential roles in peptidoglycan synthesis. In some clostridiae and mycobacteria, such as Mycobacterium tuberculosis, they are nevertheless responsible for the major peptidoglycan cross-linking pathway. L,D-Transpeptidases are thus considered as appealing new targets for the development of innovative therapeutic approaches. Carbapenems are currently investigated in this perspective as they are active on extensively drug-resistant M. tuberculosis and represent the only β-lactam class inhibiting L,D-transpeptidases. The molecular basis of the enzyme selectivity for carbapenems nevertheless remains an open question. Here we present the backbone and side-chain (1)H, (13)C, (15)N NMR assignments of the catalytic domain of Enterococcus faecium L,D-transpeptidase before and after acylation with the carbapenem ertapenem, as a prerequisite for further structural and functional studies.
    Biomolecular NMR Assignments 08/2013; 8(2). DOI:10.1007/s12104-013-9513-3 · 0.76 Impact Factor
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    • "The latter feature is modulated by secondary catalytic reactions that lead to elimination of a portion of the drug molecules prior to hydrolysis of the thioester bond. These kinetic analyses in combination with recent determination of the structures of E. faecium Ldtfm [24] and M. tuberculosis LdtMt2 [25], [26] acylated by a carbapenem or in complex with a peptidoglycan fragment [15] will pave the way for optimization of the β-lactam scaffold. "
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    ABSTRACT: 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 C(5)-C(6) 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.
    PLoS ONE 07/2013; 8(7):e67831. DOI:10.1371/journal.pone.0067831 · 3.23 Impact Factor
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