Analysis of the catlytic role of Lys224 in metallo-β-lactamase BcII from Bacillus cereus

PAOLINELLI, MARCOS; TIONI, MARIANA; VILA, ALEJANDRO

Montevideo

Conferencia; 6th International Conference of Biological Physics; 2007

Metallo-β-lactamases (MβLs) are responsible for one of the main mechanisms of bacterial resistance to β-lactam antibiotics. There is currently no therapeutically useful inhibitor against this class of enzymes. This fact reveals the importance of studying in detail their catalytic mechanism and the substrate binding mode.The catalytic mechanism used by MβLs to hydrolyse β-lactams is not fully understood but previous studies, including mutagenesis and crystallography with β-lactam derivatives haveshown that Lys 224 is implicated in substrate binding and catalysis. This residue lies in the active site but is not directly involved in metal coordination.To examine the proposed role for this residue, well conserved among different MβLs, we obtained several mutants of MβL BcII in position 224. BcII is the Mβls isolated from Bacillus cereus, an opportunistic pathogen. This MβL binds two Zn(II) ions in its active site and posseses a wide substrate spectrum that includes penicillins, cephalosporins and carbapenems. Using a PCR-based mutagenesis approach, we replaced Lys 224 with Ala, Asp and Arg. We obtained the steady-state kinetic parameters for the mutants against different β-lactams, we studied their metal content and explored the active site structure by Co(II)-substitution. Our results show that the metal content of the mutants is not affected, nor is the catalytic efficiency markedly compromised, with the greatest effect being a 2-order decrease relative to wild type. This is in contrast with similar experiments performed on other MβLs and indicates that in BcII, this position is not essential for substrate binding or catalysis