Structures of free and inhibited forms of theL,D-transpeptidase Ldt_Mt1fromMycobacterium tuberculosis

Abstract

The modelling of peptidoglycan is responsible for key cellular processes in Mycobacterium tuberculosis such as cell growth, division and resuscitation from dormancy. The structure of M. tuberculosis peptidoglycan is atypical since it contains a majority of 3,3 cross-links synthesized by L,D-transpeptidases that replace the 4,3 cross-links formed by the D,D-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these L,D-transpeptidases and in combination with clavulanic acid are bactericidal against extensively drug-resistant M. tuberculosis. Here, crystal structures of the L,D-transpeptidase LdtMt1 from M. tuberculosis in a ligand-free form and in complex with the carbapenem imipenem are reported. Elucidation of the structural features of LdtMt1 unveils analogies and differences between the two key transpeptidases of M. tuberculosis: LdtMt1 and LdtMt2. In addition, the structure of imipenem-inactivated LdtMt1 provides a detailed structural view of the interactions between a carbapenem drug and LdtMt1. By providing the key interactions in the binding of carbapenem to LdtMt1, this work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antitubercular agents against drug-resistant M. tuberculosis.

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

“…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.…”

“…A single transmembrane domain at the N terminus anchors the protein to the plasma membrane, while the extracellular domain with the catalytic site protrudes into the peptidoglycan layer. A crystal structure of Ldt Mt1 and a detailed description of its active site and its interaction with imipenem have also been recently reported (23).…”

“…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.…”