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

VILA, A J; GIANNINI, ESTEFANÍA; MARÍA-ROCÍO MEINI; GONZALEZ, L

Boston

Simposio; National meeting of the American Chemical Society; 2015

American Chemical Society

Metallo-β-lactamases (MBLs) represent the most recent generation ofcarbapenem-hydrolyzing enzymes, providing resistance to pathogenic bacteriaagainst these ?last-resort? antibiotics. MBLs from different bacteria display avery low sequence identity, and are hence divergent proteins, which,nonetheless, share a fully conserved zinc binding motif in the active site. Asa result, Zn(II) ligands and substrate-recognizing residues are fullyconserved. Nature fine tunes the substrate profile by mutations in Outersphere residues, resulting in different resistance patterns.Wehave explored the role of Outer sphere residues in MBLs. Unique Outer sphere residues are present inSPM-1, an MBL found only in Pseudomonas strains, which endow this enzyme with aunique substrate profile against anti-pseudomonas antibiotics. In addition,these residues are able to optimize the catalytic efficiency, the enzyme stability and the Zn(II) bindingfeatures in this particular bacterial host.Directedevolution experiments on a selected MBL give rise to an optimized enzyme with100-fold enhanced antibiotic resistance. This enhancement is exclusively due toOuter 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 MBLsduring evolution, as expected for enzymes expected to work under Zn(II)starvation conditions.