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Grafting a new metal ligand in the cocatalytic site of B. cereus metallo-ß-lactamase: Structural flexibility without loss of activity

¹ Molecular Biology Division, IBR (Instituto de Biología Molecular y Celular Rosario) CONICET (Consejo Nacional de Investigaciones Cientificas y Técnicas) and Biophysics Section, Department of Biological Chemistry, University of Rosario, Suipacha 531, S2002LRK Rosario, Argentina
² Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
³ Centro Regional de Estudios Genomicos, La Plata, Argentina

Reprint requests to: Alejandro J. Vila, IBR and Biophysics Section, Department of Biological Chemistry, University of Rosario, Suipacha 531, S2002LRK Rosario, Argentina; e-mail: vila{at}arnet.com.ar; fax: +54-341-4390465.

Metallo-ß-lactamases are zinc enzymes able to hydrolyze the four-membered ring of ß-lactam antibiotics, representing one of the latest generations of ß-lactamases. These enzymes belong to the zinc metallo-hydrolase family of the ß-lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild-type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.

Keywords: Metallo-ß-lactamase; metal site engineering; zinc enzymes; metal substitution; spectroscopy

Abbreviations: BcII, metallo-ß-lactamase from Bacillus cereus • L1, metallo-ß-lactamase from Stenotrophomonas maltophilia • ROO, rubredoxin-oxygen oxydoreductase from Desulfovibrio gigas • HCAII, human carbonic anhydrase II • BSA, bovine serum albumin • EDTA, ethylenedinitrilotetraacetic acid • XANES, X-ray absorption near-edge structure • AAS, atomic absorption spectroscopy

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