Mutations in R220 in PER-2 beta-lactamase resulting in a decreased susceptibility to inhibitors also impact in the catalytic activity towards substrates

M RUGGIERO ; E SAUVAGE; MF TRONCOSO; L CURTO; M GALLENI; P POWER; G GUTKIND

San Francisco

Congreso; 52nd International Conference on Antimicrobial Agents and Chemotherapy (ICAAC); 2012

Background: PER β-lactamases are a narrow group of ESBLs presenting high catalytic efficiencies towards oxyimino-cephalosporins, easily inhibited by mechanism-based inhibitors such as clavulanate. We have recently observed that mutations occurring at R220 have an impact on the inhibition susceptibility of PER-2 against clavulanate, equivalent to the role of R244 in inhibitor resistant TEM (IRT) variants. We evaluated the influence of mutations in PER-2R220 in the kinetic behavior towards different β-lactam antibiotics.   Methods: Wild-type blaPER-2 gene was amplified by PCR from an E. coli transconjugant strain (TC9). Derived mutants in R220 were generated by PCR-based site-directed mutagenesis. All amplicons were cloned in pET28a vector, b-lactamases purified by affinity chromatography, and histidine-tags eliminated with thrombin. Kinetic parameters to selected β-lactams and inhibitors were determined.   Results: According to theoretical modelizations of wild-type PER-2 and derived mutants, the environment of residues at position 220 might have influence on both the inhibition by mechanism-based inhibitors and also the activity towards several compounds. Kinetic data show that the increase in the IC50 values for inhibitors displayed by R220 mutants correlates with increments in the Km values for tested substrates. However, while kcat for penicillins also increased resulting in similar catalytic efficiencies, kcat for cephalosporins diminished giving kcat/Km values up to 10-fold lower. Also, among the tested antibiotics, it seems that this behavior is more evident for cephalosporins than penicillins.   Conclusions: Modifications in PER-2R220 not only affect the susceptibility to inhibitors but also seem to impact on the catalytic behavior towards several antibiotics. It would be interesting to evaluate the actual role of R220 in this behavior, and the influence of other important residues not only in the activity but also in structural properties.