Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity

Abstract

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldtfm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldtfm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldtfm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldtfm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldtfm acylation was followed by rupture of the C5–C6 bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldtfm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

Discovery of New Anti-Schistosomal Hits by Integration of QSAR-Based Virtual Screening and High Content Screening

“…For both compounds, the PPMs picked up the positive contributions of the basic core structure of cephalosporines, more specifically carbon 8 and nitrogen 5 of β -lactam ring, and partially positive contribution of 1-methyl-5-tetrazolethione core for inhibition of Sm TGR. On the basis of these results, we suggest that inhibition of Sm TGR by cephalosporins may occur via a mechanism similar to proposed by Triboulet and colleagues, 56 i.e., a nucleophilic attack of Cys or Sec on β -lactam carbonyl carbon, with formation of a tetrahedral intermediate, which collapses with β -lactam ring opening by N5–C8 bond fission. Then, the acyl-enzyme intermediate could hydrolyze or react further, with expulsion of the 1-methyl-5-tetrazolethione from carbon 3 generating a reactive methylene that could be trapped by other thiolate or selenoate (Supporting Information, Figure S2B).…”

Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic

“…Ldt Mt1 inactivation is a two-step reaction involving the reversible formation of a tetrahedral intermediate (oxyanion [EI ox ]), followed by irreversible acylation of the active-site cysteine (35,36). The resulting acyl enzyme, EI*, is hydrolyzed at a low rate.…”

In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by l , d -Transpeptidases and Inactivation of These Enzymes by Carbapenems

“…Fluorescence kinetics indicated that Ldt Mt1 , Ldt Mt2 , Ldt Mt3 , and Ldt Mt4 , but not Ldt Mt5 , were inactivated by carbapenems (Table 3), in agreement with mass spectrometry analyses (Table 2). The rate constants k 1 and k 2 for formation of the oxyanion and acyl enzyme, respectively, were determined by spectrofluorometry according to previously described procedures (20,23). The k 2 /K app ratio provides an evaluation of the overall efficacy of the reaction based on estimates of the disappearance of free enzyme.…”