Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity

• Published in: PloS one 2013-07, Vol.8 (7), p.e67831-e67831
• Main Authors: Triboulet, Sébastien, Dubée, Vincent, Lecoq, Lauriane, Bougault, Catherine, Mainardi, Jean-Luc, Rice, Louis B, Ethève-Quelquejeu, Mélanie, Gutmann, Laurent, Marie, Arul, Dubost, Lionel, Hugonnet, Jean-Emmanuel, Simorre, Jean-Pierre, Arthur, Michel
• Format: Article
• Language: English
• Subjects: Acylation; Amides; Ampicillin; Ampicillin - chemistry; Antibacterial activity; Antibiotics; Bacteria; Bacterial infections; Bacterial Proteins; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; beta-Lactam Resistance; Biochemistry, Molecular Biology; Biology; Carbonyls; Catalysis; Ceftriaxone; Ceftriaxone - chemistry; Cephems; Coordination compounds; Crosslinking; Deactivation; Drug resistance; Enterococcus faecium; Enterococcus faecium - chemistry; Enterococcus faecium - enzymology; Enzymes; Ertapenem; Fluorescence; Hydrolysis; Imipenem; Imipenem - chemistry; Inactivation; Kinetics; Life Sciences; Mass spectrometry; Mass spectroscopy; Medicine; Minimum inhibitory concentration; Mycobacterium tuberculosis; NMR; Nuclear magnetic resonance; Optimization; Penicillin; Penicillin-binding protein; Peptides; Peptidoglycans; Peptidyl Transferases; Peptidyl Transferases - antagonists & inhibitors; Peptidyl Transferases - chemistry; Reaction kinetics; Recombinant Proteins; Recombinant Proteins - chemistry; Serine; Streptomyces; Structural Biology; Structure-Activity Relationship; Substrate Specificity; Tuberculosis; β-Lactam antibiotics
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0067831

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 (Ldt(fm)) 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, Ldt(fm) 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 Ldt(fm) 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 Ldt(fm) inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt(fm) acylation was followed by rupture of the C(5)-C(6) 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 Ldt(fm) 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.