Characterization of a carbapenem hydrolyzing metallo-b-lactamases from Chryseobacterium meningosepticum

MORÁN BARRIO, JORGELINA; GONZALEZ, J. M.; LIMANSKY, A. S.; VIALE, A. M.; VILA, A. J.

Angra dos Reis, Brazil.

Congreso; First Latin American Protein Society Meeting.; 2004

Latin American Protein Society

b-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 zinc-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 ab/ba folding shared with the enzymes belonging to the superfamily of MbLs. GOB exhibits 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. In contrast to other subclass B3 metallo-b-lactamases, GOB is isolated with only one equivalent of zinc per mole of protein, exhibiting a Zn(II)-dependence of b-lactamase activity in vitro. As expected, the highest catalytic efficiencies were determined for carbapenems (imipenem and meropenem). These data further highlight the structural and functional diversity of MbLs from different sources. GOB. In the primary sequence we identified the metal-binding motifs and a predicted ab/ba folding shared with the enzymes belonging to the superfamily of MbLs. GOB exhibits 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. In contrast to other subclass B3 metallo-b-lactamases, GOB is isolated with only one equivalent of zinc per mole of protein, exhibiting a Zn(II)-dependence of b-lactamase activity in vitro. As expected, the highest catalytic efficiencies were determined for carbapenems (imipenem and meropenem). These data further highlight the structural and functional diversity of MbLs from different sources. GOB. In the primary sequence we identified the metal-binding motifs and a predicted ab/ba folding shared with the enzymes belonging to the superfamily of MbLs. GOB exhibits 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. In contrast to other subclass B3 metallo-b-lactamases, GOB is isolated with only one equivalent of zinc per mole of protein, exhibiting a Zn(II)-dependence of b-lactamase activity in vitro. As expected, the highest catalytic efficiencies were determined for carbapenems (imipenem and meropenem). These data further highlight the structural and functional diversity of MbLs from different sources.