title: Trapping and characterization of a reaction intermediate in carbapenem hydrolysis by B. cereus metallo-beta-lactamase.
abstract: Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.
authors.affiliation: Instituto de Biologia Molecular y Celular de Rosario and Biophysics Section, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Mariana F
authors.initials: MF
authors.lastname: Tioni
authors.forename: Leticia I
authors.initials: LI
authors.lastname: Llarrull
authors.forename: Andrés A
authors.initials: AA
authors.lastname: Poeylaut-Palena
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Martí
authors.forename: Miguel
authors.initials: M
authors.lastname: Saggu
authors.forename: Gopal R
authors.initials: GR
authors.lastname: Periyannan
authors.forename: Ernesto G
authors.initials: EG
authors.lastname: Mata
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Murgida
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology*; D015780:Carbapenems / Q000737:chemistry* / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman; D013237:Stereoisomerism; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D015780:Carbapenems; D003035:Cobalt; D001618:beta-Lactamases