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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...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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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. |
format | Online Article Text |
id | pubmed-3701632 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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