Protonation state and substrate binding to B2 metallo-b-lactamase CphA from Aeromonas hydrophila

SIMONA, F.; MAGISTRATO, A.; VERA, M.; GARAU, G.; ALEJANDRO JOSE VILA; CARLONI, P

Proteins

Año: 2006

0887-3585

The zinc enzymes metallo b -lactamases counteract the benecial actionof b -lactam antibiotics against bacterial infections. These enzymes hydrolyzeb -lactam rings. To understand structure/function relationships ona representative member of this class, the B2 Mb L CphA, we have investigatedthe H-bond pattern at the Zn site and substrate binding mode by molecularsimulation methods. Extensive QM calculations at the DFT-BLYP levelon 11 models of the protein active site, along with MD simulations of theprotein in aqueous solution, allow us to propose two plausible protonationstates for the unbound enzyme, which are probably in equilibrium. Dockingprocedures along with MD simulations and QM calculations suggest that inthe complex between the enzyme and its substrate (biapenem), the latter isstable in only one of the two protonation states, in addition it exhibits twodifferent binding modes, of which only one agrees with previous proposals.In both cases, the substrate is polarized as in aqueous solution. We concludethat addressing mechanistic issues on this class of enzymes requires acareful procedure to assign protonation states and substrate docking modes.‡