Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Hydrolysis Reaction

BACKGROUND: A general mechanism has been proposed for metallo β-lactamases (MβLs), in which deprotonation of a water molecule near the Zn ion(s) results in the formation of a hydroxide ion that attacks the carbonyl oxygen of the β-lactam ring. However, because of the absence of X-ray structures that show the exact position of the antibiotic in the reactant state (RS) it has been difficult to obtain a definitive validation of this mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We have employed a strategy to identify the RS, which does not rely on substrate docking and/or molecular dynamics. Starting from the X-ray structure of the enzyme:product complex (the product state, PS), a QM/MM scan was used to drive the reaction uphill from product back to reactant. Since in this process also the enzyme changes from PS to RS, we actually generate the enzyme:substrate complex from product and avoid the uncertainties associated with models of the reactant state. We used this strategy to study the reaction of biapenem hydrolysis by B2 MβL CphA. QM/MM simulations were carried out under 14 different ionization states of the active site, in order to generate potential energy surfaces (PESs) corresponding to a variety of possible reaction paths. CONCLUSIONS/SIGNIFICANCE: The calculations support a model for biapenem hydrolysis by CphA, in which the nucleophile that attacks the β-lactam ring is not the water molecule located in proximity of the active site Zn, but a second water molecule, hydrogen bonded to the first one, which is used up in the reaction, and thus is not visible in the X-ray structure of the enzyme:product complex.