Mechanistic studies on the mononuclear Zn(II)-containing metallo-β-lactamase ImiS from Aeromonas sobria †

Bacterial resistance to antibiotics is a growing clinical concern (1, 2). Zn(II)-containing β-lactamases (metallo-β-lactamases, MβL’s) contain 1–2 moles of Zn(II) per mole of enzyme, hydrolyze all known cephalosporins, carbapenems and penicillins, are not inhibited by clavulanic acid and other classical β-lactamase inhibitors, and have no known clinically-useful inhibitor towards them (3, 4). Previous studies have shown that there is significant structural and mechanistic diversity among the MβL’s, leading to the grouping of the enzymes into three distinct subclasses: B1, B2, and B3 (5, 6). Sequence identity ranges from 25–40% between members in one subclass and from 10–20% between members in different subclasses. Subclass B1 enzymes have been found in strains of Bacillus, Bacteroides, Pseudomonas, Serratia, and Chryseobacterium, and subclass B3 enzymes have been found in strains of Stenotrophomonas, Legionella, Fluoribacter, Janthinobacterium and Caulobacter (3, 4). Enzymes from the B1 and B3 subclasses have broad substrate profiles and require two Zn(II) ions for maximal enzymatic activity (3). In contrast, subclass B2 enzymes have a relatively narrow substrate profile, hydrolyzing carbapenems almost exclusively (3) and exhibit maximal activity when bound to only 1 Zn(II) (7). In fact, the binding of a second Zn(II) ion is inhibitory (7). These carbapenemases are produced by various species of Aeromonas. Aeromonads are enteric, anaerobic, Gram-negative pathogens that cause a range of symptoms from mild diarrhea to acute gastroenteritis in humans (8), and Aeromonas species have been found in wound infections (9). One alarming characteristic of Aeromonads is that they are water-borne and typically are present in purified, drinking water (10). To date there is no crystal structure available for ImiS, but the crystal structure of another subclass B2 enzyme, CphA from Aeromonas hydrophila, has been recently reported (11). This structure showed Zn(II) bound to His263, Cys221, Asp120, and a water; this site is the consensus Zn2 site in MβL’s (3). Recent spectroscopic studies have demonstrated that Zn(II)/Co(II) preferentially binds to this same site in ImiS (12). Previous biochemical, kinetic, and inhibition studies have suggested significant structural and mechanistic differences among the different group B β-lactamases (3, 4, 13). These results suggest that one inhibitor may not be effective in treating all infections caused by bacteria that produce a metallo-β-lactamase. Toney and Moloughney recently reviewed the literature on inhibitors of MβL’s (13), and most of these inhibitors are effective against only one or two of the enzymes. This is not surprising since, with few exceptions (14–16), the design of most of the inhibitors was guided by studies on only one enzyme. To address this problem, we are currently characterizing a metallo-β-lactamase from each of the group B subclasses (CcrA, ImiS, and L1) in an effort to identify common structural/mechanistic properties of the enzymes towards which a single inhibitor can be designed. While extensive mechanistic studies have been reported on B1 and B3 MβL’s (17–24), there is little known about the mechanism of a B2 MβL. Herein, we describe the first detailed mechanistic studies on a B2 MβL, specifically ImiS from A. veronii bv. sobria.