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

Abstract

With multi-drug resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug-targets are becoming an urgent need. Traditional β-lactam antibiotics that disrupt the D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on β-lactam insensitive 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 LdtMt2 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 LdtMt2 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 for the development of novel drugs targeting this validated yet unexploited enzyme.

Nonclassical Transpeptidases of Mycobacterium tuberculosis Alter Cell Size, Morphology, the Cytosolic Matrix, Protein Localization, Virulence, and Resistance to β-Lactams

“…The crystal structure of Ldt Mt2 was recently reported by four independent groups (19)(20)(21)(22). These studies also report molecular interactions with the substrate and the carbapenem class of drugs and the mechanism of catalysis by Ldt Mt2 .…”

“…Although ␤-lactams are rarely used for treatment of M. tuberculosis infection, recent reports demonstrate that the carbapenem subclass of ␤-lactams are effective against a range of M. tuberculosis strains (44)(45)(46). Although certain carbapenems have been demonstrated to bind to and inhibit L,D-transpeptidase activity (19,(21)(22)(23), it is not known if their M. tuberculosis growthinhibitory activity is a result of inhibition of specific or multiple transpeptidases. The in vivo study demonstrated that loss of ldt Mt1 and ldt Mt2 makes M. tuberculosis uniquely susceptible when exposed to a combination of amoxicillin-clavulanate and vancomycin.…”

“…While biochemical properties and the contribution of Ldt Mt2 to the physiology of M. tuberculosis have been reported (16,(19)(20)(21)(22), studies of Ldt Mt1 have been limited to biochemical characterization (6,23). In this study, for the first time, we have generated and studied M. tuberculosis strains lacking Ldt Mt1 and both Ldt Mt1 and Ldt Mt2 and describe the cellular phenotypes associated with the loss of these L,D-transpeptidases.…”

“…Despite the significance of D,D-transpeptidases, which serve as targets to the most widely used class of antibiotics, the study of the relevance of L,D-transpeptidases to the physiology of the cell and susceptibility to drugs has only recently begun. Within 6 months following the first report describing the crystal structure and mechanism of L,Dtranspeptidation by Ldt Mt2 (19), four additional reports describing structural details of L,D-transpeptidases of M. tuberculosis were published (20)(21)(22)(23).…”

Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

“…It has been demonstrated that ␤-lactams interfere with peptidoglycan biosynthesis and metabolism in multiple ways and eventually results in cell death (17,19). Among ␤-lactams, carbapenems and penems are able to bind and inhibit activities of D,D-transpeptidases, L,D-transpeptidases, and D,D-carboxypeptidases (6,(20)(21)(22)(23)(24)(25)(26). This versatile property of (carba)penems to target multiple enzymes prompted the current study to test the clinical utility of commercially available (carba) penems in M. tuberculosis and M. abscessus isolates from patients.…”