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

AuthorsPérez-Llanera, Francisco José; Cartelle, Mónica; Mallo, Susana; Beceiro, Alejandro; Perez, Astrid; Villanueva, Rosa; Romero, Antonio ; Bonnet, Richard; Bou, Germán
β-lactamase inhibition
cefotaxime hydrolysis
Issue DateApr-2008
PublisherOxford University Press
CitationJournal of Antimicrobial Chemotherapy 61(4):792-797(2008)
AbstractObjectives 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 β-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 blaCTX-M-1 gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of β-lactams alone and in combination with β-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (kcat, Km, kcat/Km) towards cefalotin and cefotaxime were determined. Clavulanic acid IC50 values were also assessed with all enzymes. Results No increase in MICs of β-lactam/β-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 β-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
Description6 páginas, 2 figuras, 3 tablas -- PAGS nros. 792-797
Publisher version (URL)http:dx.doi.org/10.1093/jac/dkn031
Appears in Collections:(CIB) Artículos
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