IN THE SEARCH FOR NATIVE GOB METALLO-b-LACTAMASE SUITABLE FOR CRYSTALLIZATION

MORÁN-BARRIO J, VILA AJ

Buzios, Rio de Janeiro, Brasil.

Congreso; VII Congreso Iberoamericano de Biofísica.; 2009

Sociedad de Biofísica de Brasil

Metallo-b-lactamases (MbLs) are bacterial zinc-dependent enzymes. These enzymes are present in the periplasmic space of gram-negative pathogenic bacteria and are capable of hydrolyzing most b-lactam antibiotics, being resistant to all clinically used inhibitors. This, and the fact that most of MbL genes have been found in mobile genetic elements raises a vital problem in the clinical settings. We have already characterized the MbL GOB from the gram-negative pathogen Elizabethkingia meningoseptica. GOB showed unique characteristics: it is the only broad spectrum mononuclear MbL characterized so far. Unfortunately, recombinant GOB expressed in the cytoplasm of E. coli was unable to crystallize, so the 3D structure is not available yet. Since the structural features that govern the catalytic activity in MbLs and the role of the metal ion content of these enzymes are still under debate, it is an important issue to obtain the crystal structure of this enzyme.In this work, we settled the conditions for obtaining native GOB from the periplasmic space of gram-negative bacteria. Different large-scale osmotic shock methods and lysozyme treatments were optimized, and the enzyme was purified to homogeneity from E. coli periplasmic fraction.