GOB: the only truly mononuclear broad spectrum zinc-â-

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

Rosario

Congreso; Sociedad Argentina de Biofisica; 2006

Metallo-â-lactamases (MâLs) 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 MâL: GOB from the opportunistic pathogen binding amino acid residues at the active site. We describe here the biochemical and biophysical characterization of a recently discovered MâL: GOB from the opportunistic pathogen â-lactamases (MâLs) 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 MâL: GOB from the opportunistic pathogenâL: GOB from the opportunistic pathogen Elizabethkingia meningoseptica [1]. MâLs related to GOB present two Zn(II) binding sites: Zn1 is coordinated by three His residues at the positions 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 known broad spectrum MâLs which are maximally active in their dinuclear 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 MâLs. 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 MâLs. is coordinated by three His residues at the positions 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 known broad spectrum MâLs which are maximally active in their dinuclear 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 MâLs. 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 MâLs. [1]. MâLs related to GOB present two Zn(II) binding sites: Zn1 is coordinated by three His residues at the positions 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 known broad spectrum MâLs which are maximally active in their dinuclear 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 MâLs. 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 MâLs. âLs which are maximally active in their dinuclear 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 MâLs.âLs.