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.

91 Reads
  • Source
    • "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
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
Show more