Structural and Functional characterization of Bacillus cereus BcII Zn2 site

GONZÁLEZ LJ; TOMATIS PE; VILA AJ

Montevideo, Uruguay

Congreso; XXXVI Annual Meeting of the Argentinean Biophysical Society. 6th International Conference of Biological Physics. 5th Southern Cone Biophysics Congress; 2007

International Conference on Biological Physics (ICBP)

Metallo-beta-lactamases (MBLs) are bacterial zinc-dependent enzymes able to hydrolyze a broad range of beta-lactam antibiotics. Their active sites are highly conserved, being able to bind up to two Zn(II) ions, known as Zn1 and Zn2. According to sequence homology MBLs can be divided into three subclasses: B1, B2 and B3. Subclass B1 B. cereus BcII enzyme contains three His residues and one OH- as Zn1 ligands, and His, Asp and Cys residues as Zn2 ligands. Up to date 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 which all 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 species suggest that the metal ion is pentacoordinated with one of the ligands being the Cys 221 residue of the Zn2 site. This result was in agreement with EXAFS of the Zn(II) enzyme and paramagnetic NMR of the Co(II) substituted enzyme, which in addition evidenced the presence of Zn2 site His and Asp residues in the coordination sphere.The activity of this mutant against several beta-lactams compounds was severely compromised 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 not discard the possibility of mono-Zn BcII as a minor active specie, provided the integrity of the vacant site is maintained.