Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp : Asn170Gln with benzylpenicillin and cephaloridine

The serine-beta -lactamases hydrolyze beta -lactam antibiotics in a reactio n that proceeds via an acyl-enzyme intermediate. The double mutation, E166D :N170Q, of the class A enzyme from Staphylococcus aureus results in a prote in incapable of deacylation. The crystal structure of this beta -lactamase, determined at 2.3 Angstrom resolution, shows that except for the mutation sites, the structure is very similar to that of the native protein. The cry stal structures of two acyl-enzyme adducts, one with benzylpenicillin and t he other with cephaloridine, have been determined at 1.76 and 1.86 Angstrom resolution, respectively. Both acyl-enzymes show similar key features, wit h the carbonyl carbon atom of the cleaved beta -lactam bond covalently boun d to the side chain of the active site Ser70, and the carbonyl oxygen atom in an oxyanion hole. The thiadolizine ring of the cleaved penicillin is loc ated in a slightly different position than the dihydrothiazine ring of ceph aloridine. Consequently, the carboxylate moieties attached to the rings for m different sets of interactions. The carboxylate group of benzylpenicillin interacts with the side chain of Gln237. The carboxylate group of cephalor idine is located between Arg244 and Lys234 side chains and also interacts w ith Ser235 hydroxyl group. The interactions of the cephaloridine resemble t hose seen in the structure of the acyl-enzyme of beta -lactamase from Esche richia coli with benzylpenicillin. The side chains attached to the cleaved beta -lactam rings of benzylpenicillin and cephaloridine are located in a s imilar position, which is different than the position observed in the E. co li benzylpenicillin acyl-enzyme complex. The three modes of binding do not show a trend that explains the preference for benzylpenicillin over cephalo ridine in the class A beta -lactamases. Rather, the conformational variatio n arises because cleavage of the beta -lactam bond provides additional flex ibility not available when the fused rings are intact. The structural infor mation suggests that specificity is determined prior to the cleavage of the beta -lactam ring, when the rigid fused rings of benzylpenicillin and ceph aloridine each form different interactions with the active site.