PMC

Localization of mutations harbored by SsoPox monovariants, αsA1, αsA6, αsB5, αsC6 and αsD6. Mutations are highlighted in red spheres using the SsoPox wild-type (PDB 3UF9) as a template.

Overview of SsoPox wild-type and variants properties. Catalytic efficiencies towards organophosphate-based pesticides are presented using a color code ranging from white to dark blue. Melting temperatures are presented using a color code ranging from red to green.

Chemical structure of substrates used in this study. The chemical structure of ethyl-paraoxon (I), ethyl-parathion (II), methyl-paraoxon (III), methyl-parathion (IV), malathion (V), chlorpyrifos (VI), diazinon (VII), fenitrothion (VIII), fensulfothion (IX), coumaphos (X), 3-oxo-C10 AHL (l) (XI), undecanoic-γ-lactone (XII) and undecanoic-δ-lactone (XIII) generated using ChemDraw software.

Activity characterization of the improved variants and bioremediation potential. (A) Phosphotriesterase catalytic efficiency comparison between wt-SsoPox and the best selected variants. Data for wt-SsoPox are from Hiblot et al.. (B) Determination of the reaction time for hydrolyzing 95% using the αsD6 variant of a solution of pesticide at 250 µM. Two enzyme-to-substrate ratios (), 10⁻² and 10⁻³, were considered.

Mutants’ active site loop 8 exhibit higher conformational flexibility. (A) Cartoon representation of the superposition of the structures (monomers) of wt-Ssopox (dark green), αsA1 (orange), αsB5 (yellow), αsD6 (green), αsC6 (blue), αsA6-CC (magenta) and αsA6-OC (cyan). (B) Smoothed ribbon representation of the superposition of the structures (monomers) of of wt-Ssopox (green), αsA1 (orange), αsB5 (yellow), αsD6 (green), αsC6 (blue), αsA6-CC (magenta) and αsA6-OC (cyan). (C) Positional distributions of normalized B-factor values (the x-axis represents residue number) for wt-SsoPox (black line), αsA1 (orange), αsB5 (yellow), αsD6 (green), αsC6 (blue), αsA6-OC (blue line) and (magenta line). A zoomed inset highlights the loop 7 and 8 sequence region.

Loop 8 conformations in improved mutants. Active site loop 8 region of mutant structures superimposed with the wt-SsoPox structure. The active site location is indicated by the presence of the bi-metallic catalytic center shown as spheres. The wt-SsoPox structure loop 8 conformation is shown as dark green cartoon. (A) αsA1′s loop 8 is shown in orange cartoon, (B) αsD6′s loop 8 is shown in green cartoon, and is not entirely visible in the electronic density maps, (C) αsC6′s loop 8 is shown in blue cartoon, (D) αsB5′s loop 8 is shown in yellow cartoon, and is not entirely visible in the electronic density maps, (E) αsA6-CC’s loop 8 is shown in magenta cartoon, (F) αsA6-OC’s loop 8 is shown in cyan cartoon.

Structure-based design approach. (A) Pipeline used for the selection of improved mutants. (B) Structural alignments of SsoPox (red) and BdPTE structures (yellow) guided the mutational design. (C) Active site superposition of BdPTE (green) and SsoPox (cyan) active sites. Residues with structural equivalent (orange sticks) in both structures were used to design mutations of SsoPox’s residues into the BdPTE corresponding residues. Positions with no structural equivalent (red sticks) were designed using modelling tools (purple sticks) to mimic the active site cavity size, shape and chemical properties of the BdPTE crystal structure model (Figure ). (D) The mutations data base consists of mutations to the BdPTE sequence when there is a structural equivalence (orange), and of mutations designed when there is no structural equivalence (red). The third set of mutations (black) adds more diversity at two selected positions, 258 and 263. (E) Once the library had been validated, primers carrying mutations were used to shuffle them and generate a gene library with random combinations of selected mutations. (F) The library was screened for paraoxonase activity to identify enzymes with improved proficiency.