GOB: the only truly mononuclear broad spectrum zinc-b-lactamase

MARIA NATALIA LISA; JORGELINA MORAN-BARRIO; JAVIER MARCELO GONZALEZ; ALISON COSTELLO; DAVID L. TIERNEY; ADRIANA LIMANSKY; ALEJANDRO VIALE; ALEJANDRO J. VILA

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

Metallo-beta-lactamases (MbLs) are Zn(II) dependent enzymes with highly conserved metal binding amino acid residues at the active site. We describe here the biochemical and biophysical characterization of a recently discovered MbL: GOB form the opportunistic pathogen Elizabethkingia meningoseptica. MbLs related to GOB present two Zn(II) binding sites: Zn1 is coordinated by three His residues at the position 116, 118 and 196, while Zn2 is ligated to residues Asp120, his121 and His263. However, GOB presents a Gln residue at the position 116 instead of a His. Spectroscopy data indicate that GOB?s active site harbors only one Zn(II) ion. On the other hand, kinetic studies indicate that GOB is a broad substrate-spectrum enzyme. This contrasts with the behavior of all other broad spectrum MbLs which are maximally active in their di-nuclear forms. In order to elucidate Zn(II) location in GOB?s active site, two point mutants were generated. Q116H-GOB presents spectral and kinetic characteristics that closely resemble those of the wt enzyme. This indicates that residue Gln116 is neither a metal ligand nor essential for activity in GOB. In contrast, D120S-GOB mutant shows a dramatic reduction in metal content and activity comparing to those of the wild-type enzyme, demonstrating its essentiality for metal binding and substrate hydrolysis. The overall results indicate that GOB harbors one Zn(II) ion in the canonical site-2, coordinated by residues D120, H121 and H263, therefore revealing a novel mononuclear Zn(II) site different from all currently known MbLs.