Metallo-beta-lactamase double mutants: two is a crowd

JAVIER MARCELO GONZALEZ; RODOLFO RASIA; JULIA CRICCO; ALEJANDRO J. VILA

San Carlos de Bariloche

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2003

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Metallo-beta-lactamases (MBLs) belong toa broad superfamily of Zn or Fe-dependent metallo-hydrolases, sharing a common ab/ba fold. While metal bindingsites? structures are highly conserved among these enzymes, their reactivity isfinely tuned by the residues in the first and second coordination spheres. Wehave changed some of these residues at the bi-zinc site of MBL BcII from B. cereus, giving rise to twodouble-mutants, namely BcII-R121H/C221S and BcII-R121H/C221D, which resemblethe coordination mode found in other members of the superfamily. These mutantswere cloned, overexpressed in E. coliand purified with high yields. The zinc content was determined by atomicabsorption spectroscopy. Protein folding was analyzed by CD spectroscopy, andthe zinc binding sites were probed with paramagnetic 1H-RMN andelectronic absorption spectroscopy on the cobalt-substituted proteins.Hydrolase activity was tested against b-lactam antibiotics and several esters. Thesemutants showed a reduction in MBL activity with respect to BcII and the simplemutants R121H, C221S and C221D; as well as additional esterase activities thatwere not detected in the wild-type enzyme.These results corroborate the evolutionary potentialof this protein scaffold and provide hints on the substrate recognition patternof these enzymes.