title: Hydrolysis of clavulanate by Mycobacterium tuberculosis β-lactamase BlaC harboring a canonical SDN motif.
abstract: Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G(132)N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10(4)-fold increase in k cat (0.41 s(-1)), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G(132)N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G(132)N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam-clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium.
authors.forename: Daria
authors.initials: D
authors.lastname: Soroka
authors.affiliation: Fonction et Architecture des Assemblages Macromoléculaires, Institut de Biologie Intégrative de la Cellule, Université Paris-Sud, UMR 9198, Orsay, France.
authors.forename: Inès
authors.initials: I
authors.lastname: Li de la Sierra-Gallay
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Triboulet
authors.affiliation: Fonction et Architecture des Assemblages Macromoléculaires, Institut de Biologie Intégrative de la Cellule, Université Paris-Sud, UMR 9198, Orsay, France.
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Compain
authors.affiliation: Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium Diseases of the Developing World (DDW), Tres Cantos Medicines Development Campus (TCMDC), GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Lluis
authors.initials: L
authors.lastname: Ballell
authors.affiliation: Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium Diseases of the Developing World (DDW), Tres Cantos Medicines Development Campus (TCMDC), GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Barros
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium jean-emmanuel.hugonnet@crc.jussieu.fr michel.arthur@crc.jussieu.fr.
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, LRMA Equipe 12, Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France Open Collaborative Model for Tuberculosis Lead Optimisation (ORCHID) Consortium jean-emmanuel.hugonnet@crc.jussieu.fr michel.arthur@crc.jussieu.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D019818:Clavulanic Acid / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000378:metabolism*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D047090:beta-Lactams; D019818:Clavulanic Acid; D001618:beta-Lactamases