title: 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 (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.
authors.affiliation: Centre de Recherche des Cordeliers, Equipe 12, Université Pierre et Marie Curie-Paris 6, UMR S 872, Paris, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Triboulet
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.forename: Lauriane
authors.initials: L
authors.lastname: Lecoq
authors.forename: Catherine
authors.initials: C
authors.lastname: Bougault
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.forename: Louis B
authors.initials: LB
authors.lastname: Rice
authors.forename: Mélanie
authors.initials: M
authors.lastname: Ethève-Quelquejeu
authors.forename: Laurent
authors.initials: L
authors.lastname: Gutmann
authors.forename: Arul
authors.initials: A
authors.lastname: Marie
authors.forename: Lionel
authors.initials: L
authors.lastname: Dubost
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Simorre
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D000215:Acylation; D000667:Ampicillin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D002443:Ceftriaxone / Q000737:chemistry*; D016984:Enterococcus faecium / Q000737:chemistry* / Q000201:enzymology; D015378:Imipenem / Q000737:chemistry*; D007700:Kinetics; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D018440:beta-Lactam Resistance
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D015378:Imipenem; D002443:Ceftriaxone; D000667:Ampicillin; D010458:Peptidyl Transferases