Molecular Characterization of a Carbapenem-Hydrolyzing beta-lactamase from Chryseobacterium meningoseptucum

JORGELINA MORAN-BARRIO; JAVIER MARCELO GONZALEZ; ADRIANA LIMANSKY; ALEJANDRO VIALE; 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

Beta-Lactamases are bacterial hydrolases able to hydrolyze the four-membered ring of b-lactam antibiotics, rendering them inefficient. According to their primary structure, Ambler has divided them into four classes (A, B, C, D). Enzymes from classes A, C and D are serine-active, whereas those belonging to class B are metal-dependent. Most of the known metallo-b-lactamases (MbLs) are chromosomally encoded, highly divergent, and have been detected in pathogenic species of clinical relevance. Their usually broad activity spectrum, which includes carbapenems, together with their resistance to the existing clinically useful inhibitors, indicate the high potential risk of their existence. From a clinical isolate of C. meningosepticum, we have amplified the gene coding for the periplasmic b-lactamase GOB. In the primary sequence we identified the metal-binding motifs and a predicted folding (ab/ba) shared with the enzymes belonging to the superfamily of MBLs. GOB displays homology with the most divergent subclass B3 MbLs, displaying mutations in residues involved in the zinc-binding motif. The coding gene was subcloned into a PET-type vector. The metallo-enzyme was then overexpressed and purified in E. coli, rendering a 31 kDa protein. The content of the divalent ion Zn2+ was analyzed and the specific activities with different substrates were determined. These data further highlight the structural and functional diversity of MbLs from different sources.