Structural Basis for the Interaction and Processing of β-Lactam Antibiotics by l,d-Transpeptidase 3 (Ldt_Mt3) from Mycobacterium tuberculosis

Abstract

Targeting Mycobacterium tuberculosis peptidoglycans with β-lactam antibiotics represents a strategy to address increasing resistance to antitubercular drugs. β-Lactams inhibit peptidoglycan synthases such as L,D-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3 → 3 cross-links. M. tuberculosis 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 Mt3 at 1.3 and 1.8 Å 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 a β-OH-butyrate or an acetyl group (C 2 H 3 O) covalently attached to the enzyme remains, an observation that strongly supports the notion that Ldt Mt3 is inactivated by β-lactams. Docking simulations with intact β-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 β-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 β-lactams by Ldt Mt3 , which may be useful for the development of additional β-lactams with potential antitubercular activity.

LD‐transpeptidases: the great unknown among the peptidoglycan cross‐linkers

“…It is noteworthy that LDTs of the same bacterium might vary in their size and domains. For instance, Mycobacterium tuberculosis five LDTs (Ldt Mt1-5 ) have 30%-50% sequence identity, and four of them are structurally well characterized [53,[55][56][57][58]. Ldt Mt2 and Ldt Mt5 are significantly longer than Ldt Mt1 and Ldt Mt3 and possess an extra domain: the bacterial immunoglobulin-like domain BIgA [55].…”

Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

“…M.tb contains several paralogs of L,D -transpeptidases, namely Ldt Mt1 , Ldt Mt2 , Ldt Mt3 , Ldt Mt4 , and Ldt Mt5 ( Gupta et al, 2010 ). Crystal structures of Ldt Mt1 ( Correale et al, 2013 ), Ldt Mt2 ( Erdemli et al, 2012 ), Ldt Mt3 ( Libreros-Zúñiga et al, 2019 ), and Ldt Mt5 ( Brammer Basta et al, 2015 ) have been solved and reported to date. All of these paralogs contain a pocket similar to the S-pocket found in Ldt Mt2 .…”

“…Among the five L,D -transpeptidase paralogs of M.tb , Ldt Mt2 plays an important role since an M.tb strain lacking the gene encoding this enzyme exhibits attenuation in virulence and exhibits enhanced susceptibility to β-lactam antibiotics ( Bianchet et al, 2017 ; Brammer Basta et al, 2015 ; Dubée et al, 2012 ; Gupta et al, 2010 ; Libreros-Zúñiga et al, 2019 ; Sanders et al, 2014 ; Schoonmaker et al, 2014 ). Additional reports have demonstrated altered and attenuated cellular physiology of M.tb in association with the loss of function of Ldt Mt1 ( Schoonmaker et al, 2014 ) and Ldt Mt5 ( Brammer Basta et al, 2015 ).…”

Allosteric cooperation in ß-lactam binding to a non-classical transpeptidase

“…The 3-3 linkages are generated by the more recently discovered enzyme class, the L,D-transpeptidases (Mainardi et al, 2005). Among the five L,D-transpeptidase paralogs of M.tb, LdtMt2 plays an important role since an M.tb strain lacking the gene encoding this enzyme exhibits attenuation of persistence and virulence (Bianchet et al, 2017;Brammer Basta et al, 2015;Dubee et al, 2012;Gupta et al, 2010;Libreros-Zuniga et al, 2019;Sanders et al, 2014;Schoonmaker et al, 2014). Additional reports have demonstrated altered and attenuated cellular physiology of M.tb in association with the loss of function of LdtMt1 (Schoonmaker et al, 2014) and LdtMt5(Brammer Basta et al, 2015).…”