Catalytic Mechanism, Evolution and Dynamic Features of Zinc Beta-lactamases: A novel perspective into antibiotic resistance

A. J. VILA

Tucumán

Conferencia; DAAD, Protein Dynamics And Reactivity; 2011

DAAD

Metallo-beta-lactamases are a novel family of zinc enzymes. They represent a clinical threat due to their ability to hydrolyze beta-lactam antibiotics, displaying a broad substrate profile. No clinically useful inhibitor is yet available for these metalloenzymes. Enzymes from different sources exhibit an outstanding diversity in metal coordination features, metal requirements for activity, and catalytic efficiencies within a common protein fold. We have explored the versatility of the metal binding capabilities and functionality beta-lactamase from B. cereus, (LII) by using site-directed mutagenesis. This enzyme is able to bind up to two zinc(II) ions, although only one of them is essential for the enzyme activity. The mutations were aimed to tune the binding capabilities and functionality of the second zinc(II) binding site. The cobalt(II) derivatives of the different mutants were studied by different spectroscopic techniques in order to elucidate the structure of the novel metal sites. Mutants on Arg91, which hydrogen bonds to a metal ligand of the second zinc(II), drastically alter the function of this metal equivalent. Engineering of new metal ligands give rise to mutants that are inhibited upon binding of the second zinc(II). Elucidation of the structure of the inhibited mutants reveals intimate features of the catalytic mechanism of the enzyme. Mutation of Cys 168 (a ligand of the second zinc(II) ion) by an Asp gives rise to an enzyme with impaired activity, that is hyperactivated at high pH. This protonation event triggers a conformational change in the active site that is not present in the native enzyme. The high pH form resembles closer the active site of wild type LII, revealing the minimal geometric requirements for an active enzyme. Comparison of these mutations with those present naturally in beta lactamases from other bacteria, confirm the versatility of the metal binding motif exploited by bacteria to develop efficient resistance mechanisms against antibiotics.