IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Structural and Functional characterization of Bacillus cereus BcII Zn2 site
Autor/es:
L.J. GONZÁLEZ, P.E. TOMATIS, D.L. TIERNEY, A.J. VILA
Lugar:
Montevideo-Uruguay
Reunión:
Congreso; 6th International Conference of Biological Physics; 2007
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
Metallo-β-lactamases (MBLs) are bacterial zinc-dependent enzymes able to hydrolyze abroad range of β-lactam antibiotics. Their active sites are highly conserved, being able to bindup to two Zn(II) ions, known as Zn1 and Zn2. According to sequence homology MBLs can bedivided into three subclasses: B1, B2 and B3. Subclass B1 B. cereus BcII enzyme contains threeHis residues and one OH- as Zn1 ligands, and His, Asp and Cys residues as Zn2 ligands. Up todate it is not clear the role of each metal binding site in catalysis.In an attempt to study the function of Zn2 site in BcII, we constructed a mutant in whichall the Zn1 ligands were replaced by Ser residues. This mutant enzyme (Zn2-BcII), as expected,was able to bind only 1 Zn(II)/protein. Electronic spectra of the Co(II)-substituted speciessuggest that the metal ion is pentacoordinated with one of the ligands being the Cys 221 residueof the Zn2 site. This result was in agreement with EXAFS of the Zn(II) enzyme andparamagnetic NMR of the Co(II) substituted enzyme, which in addition evidenced the presenceof Zn2 site His and Asp residues in the coordination sphere.The activity of this mutant against several β-lactams compounds was severelycompromised compared to the wild type enzyme (3-4 orders of magnitude lower). Interestingly,previous studies on a mono-Zn1 BcII mutant showed a similar reduction in catalytic efficiency,indicating that the wild type enzyme requires the integrity of both metallic sites to be active.This implies that it is essential the presence of both metal ions at the same time, but does notdiscard the possibility of mono-Zn BcII as a minor active specie, provided the integrity of thevacant site is maintained.