Role of Zinc Content on the Catalytic Efficiency of B1 Metallo β-Lactamases

Abstract

Metallo β-lactamases (MβL's) are enzymes naturally evolved by bacterial strains under the evolutionary pressure of β-lactam antibiotic clinical use. They have a broad substrate spectrum and are resistant to all the clinically useful inhibitors, representing a potential risk of infection if massively disseminated. MβL's scaffold is designed to accommodate one or two zinc ions able to activate a nucleophilic hydroxide for the hydrolysis of the β-lactam ring. The role of zinc content on binding and reactive mechanism of action has been the subject of debate and still remains an open issue despite the large amount of data acquired. We report herein a study of the reaction pathway for binuclear CcrA from Bacteroides fragilis using density functional theory based quantum mechanicsmolecular mechanics dynamical modeling. CcrA is the prototypical binuclear enzyme belonging to B1 MβL family, which includes several harmful chromosomally-encoded and transferable enzymes. The involvement of a second zinc ion in the catalytic mechanism lowers the energetic barrier for β-lactam hydrolysis, preserving the essential binding features found in mononuclear B1 enzymes (BcII from Bacillus cereus) while providing a more efficient single-step mechanism. Overall, this study suggests that uptake of a second equivalent zinc ion is evolutionary favored.

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

“…The shorter bond length in the former case is consistent with this ligand being a hydroxide and with the idea that the 3H site is responsible for lowering the pK a of a water molecule, thus being responsible for nucleophile activation (57). The role of CϭO polarization by this same zinc ion is not supported by QM/MM calculations and by different docking studies, which reveal that the ␤-lactam bond may not directly bind to the metal ion (60,61).…”

“…Substrate docking studies to the active site of B1 and B3 lactamases suggest that the attacking nucleophile in the dinuclear forms is the bridging water/OH Ϫ ligand (13,14,28,36,60). This moiety is asymmetrically positioned with respect to the two metal ions, lying closer (1.9 -2.1 Å) to the metal ion in the 3H site than to the Zn(II) ion in the DCH or DHH sites (2.1-3.1 Å) (13,14,28).…”

Common Mechanistic Features among Metallo-β-lactamases

“…In addition, our recent molecular simulation study (17) pointed to the existence of an alternative conformation of the Henry-Michaelis complex, in which a water molecule replaces Bia substrate as the fourth zinc ligand (ES2 complex in the bottom panel of Fig. 1), similar to the reactant state found in B1 M␤Ls (21,22).…”

“…As in the B1 M␤Ls enzymes (21,22), the first step of the reaction was studied by pulling the catalytic Wat1 oxygen toward the ␤-lactam carbonyl carbon (C-7). d O-C-7 (the distance between O at Wat1 and C-7 at Bia) (Figs.…”

“…This choice is similar to those used in other studies (21,22,28) and, to a first approximation, is the natural one to describe the direct nucleophilic attack. d O-C-7 was progressively shortened until the formation of the transition state (TS1) (at 1.7 Å).…”