Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by B. cereus Metallo-β-lactamase

Abstract

Metallo-β-lactamases hydrolyze most β-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 both in 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 β-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, the identification of this intermediate could be exploited as a first step towards the design of transition state based inhibitors for all three classes of metallo-β-lactamases.

Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

“…The effect of this mutation on the rate-limiting step of the reaction allows us to propose a strategy for designing a mechanism-based inhibitor with an evolutionary perspective. Recent mechanistic studies suggest that intermediate stabilization by the Zn2 site is essential in M␤L catalysis (33,34). Our results show that optimization of the chemistry in the active site can still be optimized by better positioning Zn2.…”

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…Although nitrocefin is not a clinically useful antibiotic, its usefulness as a probe of the catalytic mechanism of M␤Ls should not be underestimated. It has been shown recently that carbapenem hydrolysis by a B1 M␤L also proceeds by means of an anionic intermediate, after C-N bond cleavage (58). This evidence points to a general role of the Zn(II) ion in the DCH/DHH site of M␤L from all subclasses, which may be targeted as a common mechanistic element for inhibitor design.…”

“…This fact highlights its amazing chemical versatility. In the case of M␤Ls, the Zn(II) ion is able to contribute to ␤-lactam hydrolysis by 1) lowering the pK a of a bound water molecule, which may act as a nucleophile, providing a high local concentration of hydroxide ions at neutral pH (37,57); 2) acting as a Lewis acid, polarizing the CϭO bond and therefore augmenting the electrophilic nature of the carbonyl carbon (57); and 3) stabilizing a negative charge in the bridging nitrogen of the lactam moiety, after C-N bond cleavage (16,17,24,36,58,59). These three roles have been invoked for the two metal binding sites in M␤Ls, but it is not clear yet which of them are essential for catalysis.…”

Common Mechanistic Features among Metallo-β-lactamases

“…Despite steady-state kinetic studies at different pH values (54) suggesting the requirement of two bound metal ions for the hydrolytic activity of the B1 M␤L BcII, it has been recently shown that BcII can be active as a mono-zinc species with the metal ion accommodated at the Zn2 site, i.e. resembling the conformation of the B2 active site (55,56). Recent studies have revealed that the B3 lactamase GOB (57) is able to act as a mononuclear enzyme with the metal ion in the putative Zn2 site, i.e.…”