CRYSTAL-STRUCTURE OF THE ZINC-DEPENDENT BETA-LACTAMASE FROM BACILLUS-CEREUS AT 1.9 ANGSTROM RESOLUTION - BINUCLEAR ACTIVE-SITE WITH FEATURES OF A MONONUCLEAR ENZYME

The structure of the zinc-dependent beta-lactamase II from Bacillus ce reus has been determined at 1.9 Angstrom resolution in a crystal form with two molecules in the asymmetric unit and 400 waters (space group P3(1)21; R-cryst = 20.8%). The active site contains two zinc ions: Zn1 is tightly coordinated by His86, His88, and His149, while Zn2 is loos ely coordinated by Asp90, Cys168, and His210. A water molecule (W1) li es between the two zinc ions but is significantly closer to Zn1 and at a distance of only 1.9 Angstrom is effectively a hydroxide moiety and a potential, preactivated nucleophile. In fact, Asp90 bridges W1 to Z n? and its location is thus distinct from that nf the bridging water m olecules in the binuclear zinc peptidases or other binuclear zinc hydr olases. Modeling of penicillin, cephalosporin, and carbapenem binding shows that all are readily accommodated within the shallow active site cleft of the enzyme, and the Zn1-bound hydroxide is ideally located f or nucleophilic attack at the beta-lactam carbonyl. This enzyme also f unctions with only one zinc ion present. The Zn1-Zn2 distances differ in the two independent molecules in the crystal (3.9 and 4.4 Angstrom) , yet the Zn1-W1 distances are both 1.9 Angstrom, arguing against invo lvement of Zn2 in W1 activation. The role of Zn2 is unclear, but the B . cereus enzyme may be an evolutionary intermediate between the mono- and bizinc metallo-beta-lactamases. The broad specificity of this enzy me, together with the increasing prevalence of zinc-dependent metallo- beta-lactamases, poses a real clinical threat and this structure provi des a basis for understanding its mechanism and designing inhibitors.