Kinetic and Spectroscopic Characterization of Bacillus cereus BcII Zn2 site

GONZÁLEZ LJ; TOMATIS PE; VILA AJ

Rosario, Santa Fe, Argentina

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

Sociedad Argentina de Biofísica

Metallo-beta-lactamases (MBLs) are 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. According to the set of metal ion ligands, 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. On the other hand, subclass B2 CphA enzyme is characterized by a putative Zn1 site with one of the His replaced by an Asn residue, and is able to hydrolyze only carbapenems, while B1 and B3 MBLs are broad-spectrum. It is well known that CphA enzyme is active only as mono-Zn2 enzyme, however, for BcII it is not clear yet the role of each metal binding site in catalysis.In an attempt to study the role 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)/equivalent. The activity against several beta-lactams compounds was severely compromised compared to the wild type enzyme (3-4 orders of magnitude lower). 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 also evidenced the presence of Zn2 site His and Asp residues as part of the coordination sphere. Spectrophotometric titration of the mutant apo-enzyme with Co(II) revealed that Cys 221 residue is more exposed, being prone to oxidation, than the wild type apo-enzyme. Together with circular dichroism studies, these results suggest that the His residues of the Zn1 site are involved in a hydrogen-bond network engaged in maintaining the active site structure in the absence of metal. 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. On the other hand, the fact that B2 enzymes are active carbapenemases as monoZn2 can be attributed either to the His116Asn substitution in the Zn1 site and to topological differences in their active sites. This means that MBLs are flexible structures, capable of providing Zn1 ion functions with subtle variations in the aminoacidic composition. As Zn2 site is occupied in all MBLs, we conclude therefore that Zn2 ion is essential for b-lactamase activity, and not cocatalityc as previously proposed. The incorporation of Zn1 ion in B1 and B3 enzymes probably reflects a more versatile and advantageous evolutionary pathway for activity optimization.