Understanding the role of a conserved Cysteine residue in B1 Metallo-beta-lactamases

JAVIER MARCELO GONZALEZ; JULIA CRICCO; ALEJANDRO J. VILA

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2006

Sociedad Argentina de Biofísica

Beta-lactamases are enzymes produced by bacteria which confer them resistance against b-lactam antibiotics. Metallo-b-lactamases (MBL) require one or two Zn(II) ions for their activity. They display a broad substrate profile, which includes penicillins, cephalosporins and carbapenems. These enzymes present similar folding and conserved active-site residues. Based on sequence homology alignments they are grouped into subclasses B1, B2 and B3.  Beta-lactamase II from Bacilluscereus (BcII) belongs to B1 MBL subclass and can bind up to two Zn(II) ions. Cys221 is a conserved residue in B1 MBLs, but not in other members belonging to a large superfamily of metalloproteins. This residue acts as metal ion ligand in the so-called Zn2 site. In order to address the role of Cys221 residue in B1 MBLs we replaced Cys221 in BcII by Asp, which is conserved amongst the MBL superfamily. The mutant enzyme C221D-BcII displays a catalytic activity comparable to the WT enzyme against penicillins, nitrocefin, cefotaxime and imipenem. The Co(II)-substituted C221D- BcII enzyme exhibits the same Vis spectrum as Co(II)-BcII. This result suggests that the coordination geometry of the active site was not altered by the introduced mutation. However, stepwise titration of apo-C221D-BcII with Co(II), followed by electronic and paramagnetic H-NMR spectroscopy, evidences that the binding of Co(II) follows an ordered sequential process. The latest results suggest that the Co(II) binding in C221D-BcII is different than in the WT enzyme. This mutant allows the formation of mono and di-cobalt species that can be identified spectroscopically. This property could be useful to investigate about the process of metal binding and activity in metallo-b-lactamase enzymes.