X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: Direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid

The clinical use of beta-lactam antibiotics combined with beta-lactamase in activators, such as clavulanate, has resulted in selection of beta-lactamas es that are insensitive to inactivation by these molecules. Therefore, ther apeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 va riants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mut ations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have charac terized the kinetic properties of the inhibition process of the wild-type T EM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam , and we report the X-ray structure for the mutant variant at 2.3 Angstrom resolution. The changes in kinetic parameters for the interactions of the i nhibitors with the wild-type and the mutant enzymes were more pronounced fo r clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant moveme nt of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, whi ch is observed in the wild-type enzyme structure, is not present in the cry stal structure of the mutant variant. Such structural changes favor the tur nover process over the inactivation chemistry for clavulanate, with profoun d phenotypic consequences. The report herein represents the best studied ex ample of inhibitor-resistant beta-lactamases.