Structural Basis for the Interaction and Processing of Β-Lactam Antibiotics by L,d-Transpeptidase 3 (ldtmt3) from Mycobacterium Tuberculosis.

Targeting Mycobacterium tuberculosis peptidoglycans with beta-lactam antibiotics represents a strategy to address increasing resistance to antitubercular drugs. beta-Lactams inhibit peptidoglycan synthases such as L,D-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3 -> 3 cross-links. M. tuber-culosis encodes five L,D-transpeptidases (Ldt(Mt1)(-5)), of which Ldt(Mt3) is one of the less understood. Herein, we structurally characterized the apo and faropenem-acylated forms of Ldt(Mt)3 at 1.3 and 1.8 A resolution, respectively. These structures revealed a fold and catalytic diad similar to those of other Ldts(Mt) enzymes, supporting its involvement in transpeptidation reactions despite divergences in active site size and charges. The Ldt(Mt3)-faropenem structure indicated that faropenem is degraded after Cys-246 acylation, and possibly only beta-OH-butyrate or an acetyl group (C2H3O) covalently attached to the enzyme remains, an observation that strongly supports the notion that Ldt(Mt3) is inactivated by beta-lactams. Docking simulations with intact beta-lactams predicted key Ldt(Mt3) residues that interact with these antibiotics. We also characterized the heat of acylation involved in the binding and reaction of Ldt(Mt3) for ten beta-lactams belonging to four different classes, and imipenem had the highest inactivation constant. This work provides key insights into the structure, binding mechanisms, and degradation of beta-lactams by Ldt(Mt3,) which may be useful for the development of additional beta-lactams with potential antitubercular activity.