On the active site of mononuclear B1 metallo β-lactamases: a computational study

Metallo-beta-lactamases (M beta Ls) are Zn(II)-based bacterial enzymes that hydrolyze beta-lactam antibiotics, hampering their beneficial effects. In the most relevant subclass (B1), X-ray crystallography studies on the enzyme from point to either two zinc ions in two metal sites (the so-called '3H' and 'DCH' sites) or a single Zn(II) ion in the 3H site, where the ion is coordinated by Asp120, Cys221 and His263 residues. However, spectroscopic studies on the B1 enzyme from . in the mono-zinc form suggested the presence of the Zn(II) ion also in the DCH site, where it is bound to an aspartate, a cysteine, a histidine and a water molecule. A structural model of this enzyme in its DCH mononuclear form, so far lacking, is therefore required for inhibitor design and mechanistic studies. By using force field based and mixed quantum-classical (QM/MM) molecular dynamics (MD) simulations of the protein in aqueous solution we constructed such structural model. The geometry and the H-bond network at the catalytic site of this model, in the free form and in complex with two common beta-lactam drugs, is compared with experimental and theoretical findings of CphA and the recently solved crystal structure of new B2 M beta L from (Sfh-I). These are M beta Ls from the B2 subclass, which features an experimentally well established mono-zinc form, in which the Zn(II) is located in the DCH site. From our simulations the epsilon epsilon I ' and delta epsilon I ' protomers emerge as possible DCH mono-zinc reactive species, giving a novel contribution to the discussion on the M beta L reactivity and to the drug design process.