Structure-function studies of arginine at position 276 in CTX-M β-lactamases

Objectives: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis. Methods: By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K-m, k(cat)/K-m) towards cefalotin and cefotaxime were determined. Clavulanic acid IC50 values were also assessed with all enzymes. Results: No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC50 values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His. Conclusions: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.