INVESTIGADORES
RUGGIERO Melina
congresos y reuniones científicas
Título:
Role of Arg220 and Thr237 of PER-2 beta-Lactamase in the Stabilization of a Hydrogen Network, Essential for Interaction with beta-Lactams
Autor/es:
M. RUGGIERO; E. SAUVAGE; L. CURTO; M. GALLENI; G. GUTKIND; P. POWER
Lugar:
Washington
Reunión:
Congreso; 54th Interscience Conference on Antimicrobial Agents and Chemotherapy; 2014
Institución organizadora:
American Society for Microbiology
Resumen:
Background: Recently, we solved the crystallographic structure of PER-2 b-lactamase at 2.2 Å. From the structure, and simulated modelizations of the enzyme in combination with b-lactams and inhibitors, we proposed the existence of an important hydrogen network stabilizing the active site, and probably having impact in the activity towards them. In this study, we analyzed the phenotypic and kinetic impact of mutations in Arg220 and Thr237 towards selected b-lactams and clavulanic acid. Methods: Wild-type blaPER-2 gene was cloned in frame at the cloning site of kanamycin resistant pK19 vector. Derived mutants in R220 and T237 were generated by PCR-based site-directed mutagenesis. Antimicrobial susceptibility was evaluated by MIC. All genes were sub-cloned in pET28a vector, b-lactamases purified by affinity chromatography, and histidine-tags eliminated with thrombin. Kinetic parameters to selected b-lactams and inhibitors were determined. Circular dichroism was performed for all enzymes at 320-250 nm (near UV) and 250-200 nm (far UV). Results: Six mutants in R220 were obtained (R220G, R220L, R220S, R220T, R220H and R220C), and one in T237 (T237A). MIC values showed that R220G mutant was the most affected towards inhibition by clavulanate (from 64 to 512 ug/ml for AMC). However, all R220 mutants displayed strong decrease in MICs for nearly all tested b-lactams, except cephamycins. 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 b-lactams, especially penicillins. Circular dichroism results suggest that none of the mutations have negative impact in the overall structure. Conclusions: 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 b-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.