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

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-lactam...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701632/
https://www.ncbi.nlm.nih.gov/pubmed/23861815
http://dx.doi.org/10.1371/journal.pone.0067831
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author 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
author_facet 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
author_sort Triboulet, Sébastien
collection PubMed
description 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.
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spelling pubmed-37016322013-07-16 Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity 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 PLoS One Research Article 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. Public Library of Science 2013-07-04 /pmc/articles/PMC3701632/ /pubmed/23861815 http://dx.doi.org/10.1371/journal.pone.0067831 Text en © 2013 Triboulet et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
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
Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title_full Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title_fullStr Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title_full_unstemmed Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title_short Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity
title_sort kinetic features of l,d-transpeptidase inactivation critical for β-lactam antibacterial activity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701632/
https://www.ncbi.nlm.nih.gov/pubmed/23861815
http://dx.doi.org/10.1371/journal.pone.0067831
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