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JAC Advance Access originally published online on February 14, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):792-797; doi:10.1093/jac/dkn031
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

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

Francisco José Pérez-Llarena1, Mónica Cartelle1, Susana Mallo1, Alejandro Beceiro1, Astrid Pérez1, Rosa Villanueva1, Antonio Romero2, Richard Bonnet3 and Germán Bou1,*

1 Servicio de Microbiología, Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain 2 Departamento de Ciencia de Proteínas, Centro Investigaciones Biológicas (CSIC), Madrid, Spain 3 Laboratoire de Bactériologie, CHU Clermont-Ferrand, Faculté de Medecine, Clermont-Ferrand, France

Received 12 April 2007; returned 30 May 2007; revised 3 November 2007; accepted 3 January 2008

* Corresponding author. Tel: +34-981-176087; Fax: +34-981-176097; E-mail: germanbou{at}canalejo.org

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 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.

Keywords: clavulanate , β-lactamase inhibition , cefotaxime hydrolysis


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