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

Background: The first line of defense by bacteria against beta-lactam antibiotics is the expression of beta-lactamases, which cleave the amide bond of the beta-lactam ring. In the reaction of biapenem inactivation by B2 metallo beta-lactamases (MbLs), after the beta-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5-C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo beta-lactamase CphA in complex with product. Methodology/Principal Findings: QM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the beta-lactam ring unprotonated. Conclusions/Significance: The calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MbLs, and suggest a possible revision of mechanisms for B2 MbLs in which the cleavage of the beta-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MbLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.