Fine tuning of the catalytic efficiency and metal binding features in metalloenzymes by Outer Sphere residues

ALEJANDRO J. VILA; ESTEFANIA GIANNINI; MARÍA ROCÍO MEINI; LISANDRO J. GONZALEZ

Boston

Simposio; National meeting of the American Chemical Society; 2015

American Chemical Society

Metallo-b-lactamases (MBLs) represent the most recent generation of carbapenem-hydrolyzing enzymes, providing resistance to pathogenic bacteria against these ?last-resort? antibiotics. MBLs from different bacteria display a very low sequence identity, and are hence divergent proteins, which, nonetheless, share a fully conserved zinc binding motif in the active site. As a result, Zn(II) ligands and substrate-recognizing residues are fully conserved. Nature fine tunes the substrate profile by mutations in Outer sphere residues, resulting in different resistance patterns.We have explored the role of Outer sphere residues in MBLs. Unique Outer sphere residues are present in SPM-1, an MBL found only in Pseudomonas strains, which endow this enzyme with a unique substrate profile against anti-pseudomonas antibiotics. In addition, these residues are able to optimize the catalytic efficiency, the enzyme stability and the Zn(II) binding features in this particular bacterial host.Directed evolution experiments on a selected MBL give rise to an optimized enzyme with 100-fold enhanced antibiotic resistance. This enhancement is exclusively due to Outer sphere residues, involved in the hydrogen bond network of Zn(II) ligands. These residues play a key role in enhancing the Zn(II) binding abilities of MBLs during evolution, as expected for enzymes expected to work under Zn(II) starvation conditions.