Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm.
Mol Microbiol. 2015 Jun 23;
Authors: Triboulet S, Bougault CM, Laguri C, Hugonnet JE, Arthur M, Simorre JP
In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by L,D-transpeptidases that form unusual 3→3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate L,D-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium L,D-transpeptidase Ldtfm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An NMR-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldtfm that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilization of the acceptor in the Ldtfm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams.
PMID: 26101813 [PubMed - as supplied by publisher]