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Protonation state and substrate binding to B2 metallo‐β‐lactamase CphA from Aeromonas hydrofila

The zinc enzymes metallo β‐lactamases counteract the beneficial action of β‐lactam antibiotics against bacterial infections, by hydrolyzing their β‐lactam rings. To understand structure/function relationships on a representative member of this class, the B2 MβL CphA, we have investigated the H‐bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT‐BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes. Proteins 2007. © 2007 Wiley‐Liss, Inc.