Article

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

ABSTRACT

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.

  • 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

    Full-text · Dataset · Jul 2015
  • Source
    • "transition phases of non-or slowly replicating persistence, which coincides with decreased antimicrobial drug susceptibility . It is the slow rate of bacterial replication that is thought to limit the effectiveness of some antimicrobial drugs especially those that target cell wall synthetic pathways (Erdemli et al., 2012). The original Wayne model with and without modifications is still commonly used to better understand the factors that influence in vitro bacterial growth and M. tuberculosis drug susceptibility (Leistikow et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: There is an urgent need to improve methods used to screen anti-tuberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high throughput platform to screen new anti-tuberculosis drugs especially those that target drug tolerant bacilli. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jan 2014 · Pathogens and Disease
  • 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 1H, 13C, 15N 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.
    Full-text · Article · Aug 2013 · Biomolecular NMR Assignments
Show more