ARG220 AND THR237 OF PER-2 b-LACTAMASE HAVE AN IMPORTANT ROLE IN THE STABILIZATION OF THE ACTIVE SITE AND THE INTERACTION WITH b-LACTAMS.

RUGGIERO, M.; CURTO L.M.; GALLENI, M; CHARLIER, P.; GUTKIND, G; SAUVAGE, E; POWER, P

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Biofísica; 2014

In this study, we analyzed the impact of mutations in Arg220 and Thr237 towards selected β-lactams and clavulanic acid, by structural and kinetic analysis. Crystal structure of PER-2 was refined to 2.20 Å (PDB entry: 4D20). Wild-type blaPER-2 gene and derived mutants in R220 and T237 were generated by site-directed mutagenesis. β-Lactamases were purified to homogeneity and kinetic parameters to selected β-lactams and inhibitors were determined. Circular dichroism was performed for all enzymes at near and far UV. Catalytic efficiencies of R220 mutants towards penicillins and cephalosporins are up to 6- and 100-fold lower than wild type, respectively, whereas T237A mutant displayed slightly higher kcat/Km values for some β-lactams, especially penicillins. Circular dichroism results suggest that none of the mutations have negative impact in the overall structure. Our results support the previously suggested role of R220 and T237 in the maintenance and stabilization of a hydrogen network, necessary for the proper interaction with β-lactams. In this regard, R220 seems to be essential for interaction with both clavulanate and cephalosporins, while T237, though also important, would probably have a secondary role in this network. The apparent structural stability of the mutants also suggests that they are prone to be selected in vivo.