GOB IS A NEW MONONUCLEAR ZINC ƒÒ-LACTAMASE WITH A NOVEL ACTIVE SITE

MORAN-BARRIO, J.; GONZALEZ, J.; LISA, M.; COSTELLO, A.; TIERNEY, D.; ADRIANA SARA LIMANSKY; VILA, A.; VIALE, A.

Rosario

Congreso; Sociedad Argentina de Investigacion de Bioquimica; 2006

Metallo-b-lactamases (MbL) are Zn(II) dependent enzymes that share a common ab/ba scaffold with highly conserved metal binding amino acid residues. We describe here the biochemical and biophysical characterization of GOB, an MbL with particular amino acid residue substitutions in the metal binding site. The gob gene was amplified from a clinical isolate of the Gram-negative pathogen Elizabethkingia meningoseptica, cloned in appropriate expression vectors, and expressed in E. coli. Expression of the complete gob coding sequence in this host resulted in a native enzyme exported to the periplasm via the Sec machinery, conferring resistance to different b-lactams. Size exclusion chromatography showed that recombinant GOB is a monomer. Kinetic studies indicated that GOB is a broad-substrate spectrum enzyme displaying maximum activity with only one equivalent of Zn(II) per molecule. This contrasts all other known broad spectrum MbLs which are maximally active in their di-nuclear forms. Spectroscopic and kinetic data obtained from the wt enzyme and mutated variants demonstrated that the canonical site 2 is essential for both metal binding and activity. The overall results thus indicate that GOB harbors one Zn(II) ion coordinated by residues D120, H121, H263 and a water molecule in the active site, therefore revealing a novel mononuclear Zn(II) site different from other related MbLs.