An insight into the second Zn site of the dinuclear BcII metallo-beta-lactamase

GONZÁLEZ LJ; TOMATIS PE; VILA AJ

Pinamar, Bs. As.

Congreso; X PABMB Congress. SAIB, XLI Annual Meeting. SAN, XX Annual Meeting; 2005

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

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 [1,2]. 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 Zn1 site with one of the His replaced by an Asn residue, and have differences in the secondary structure surrounding the active site. It is well known that CphA enzyme is active only as mononuclear enzyme, however, for BcII it is not clear yet the role of each metal binding site in catalysis [3,4].In an attempt to study the role of Zn2 site in BcII, we constructed a mutant (using the “Megaprimer” method) in which all the Zn1 ligands were replaced by Ser residues. This mutant enzyme (Zn2-BcII), was able to bind only 1 Zn(II)/mole. The activity against several beta-lactams compounds (figure 3) was severely compromised compared to the wild type enzyme. Electronic spectra of the Cu(II) and Co(II)-substituted species suggest that the metal ion is penta- or hexacoordinated  with one of the ligands being a Cys residue. Paramagnetic NMR spectra of the Co(II)-substituted enzyme allow us to confirm that the  metal ion is coordinated to ligands of the Zn2 site. Circular Dichroism results suggest a structural role for the Zn(II) ion. References: 1-Orellano, Girardini, Cricco, Ceccarelli and Vila. Biochemistry. 1998 Jul 14;37(28):10173-80. 2-Cricco and Vila. Curr Pharm Des. 1999 Nov;5(11):915-27. Review. 3-Rasia and Vila. Biochemistry. 2002 Feb 12;41(6):1853-60. 4-Garau, Bebrone, Anne, Galleni, Frere, Dideberg. J Mol Biol. 2005 Jan 28;345(4):785-95.