Abstract
BackgroundA 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 FindingsWe 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/SignificanceThe 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.
Highlights
Class B b-lactamases are metallo-enzymes requiring one or two Zn2+ ions for activity [1]
Driving the Reaction Backward We studied the reaction of biapenem hydrolysis by CphA from
The structure of N220G CphA (,K the activity of wild type) was determined by Garau et al ([14], PDB entry 1X8I) in complex with a form of hydrolyzed biapenem that has undergone a molecular rearrangement [14,22] such that oxygen atom O62 forms a 6-membered ring (N4-C5-C6-C61-O62-C3) that replaces the original b-lactam ring (Figure 1C). While this bicyclic compound is the product of a minor pathway in the enzyme active site [43,44], in a prequel to this study [44] we have shown that it can be formed at a higher rate in solution from a spontaneous cyclization of hydrolyzed biapenem, after which it binds back to the enzyme and acts as a product inhibitor [22]
Summary
Class B b-lactamases are metallo-enzymes requiring one or two Zn2+ ions for activity [1]. MbLs are evolving rapidly and becoming progressively more effective and specialized against different antibiotics [5,6,7]; no clinically relevant inhibitors of these enzymes are yet available [2,8]. Based on their sequence heterogeneity MbLs have been grouped into three subclasses (B1 to B3) [1]. 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
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