Fig 1.
Cal-A was treated in three segments for mutagenesis.
Parts 1 (encoding the N-terminal 210 residues), 2 (encoding residues 211 to 350) and 3 (C-terminal residues 351 to 446 plus 6His-tag) were individually randomized and recombined with wild-type or mutated parts. Parts 1 and 3 include residues treated by NDT saturation mutagenesis (Tyr93, Tyr183 and Phe 431), shown in magenta. The catalytic triad (Ser184, Asp334, His366) is shown in yellow. Image generated using PDB coordinates 2VEO [29].
Table 1.
Library generation and assessment of library quality.
Table 2.
Discriminative variants obtained upon screening the Cal-A libraries against short- and long-chain triglycerides.
Fig 2.
Short- vs long-chain hydrolytic activity in point substituted libraries of Tyr93, Tyr183 and Phe431.
Variants were screened for halo formation around colonies spotted on solid medium. Hydrolysis of the short-chain tributyrin (Trib) or long-chain olive oil (Olive) were rated as active (green checkmark) or inactive (red ‘Χ’).
Fig 3.
Activity level of the discriminative variants towards short and long chain triglycerides.
A: Variants selected from library Random 2. B: Variants selected from libraries Random Tot and Random Rec, combined. Left panels, short-chain activity (hydrolysis of tributyrin): gradient from light yellow (low activity) to orange (high activity). Right panels, long-chain activity (hydrolysis of olive oil): gradient from light (low activity) to dark blue (high activity). Inactive variants coloured as background (gray). Wild-type activity corresponds to shade iii. Where more than one discriminative variant was mutated at the same position, the position is colored according to the variant having the highest activity. A PEG-4 molecule is shown in black spheres, crystallized inside the putative tunnel [22, 29]. Libraries Random Tot and Random Rec are individually represented in S1 Fig. Libraries Random 1 and Random 3 did not yield discriminative variants and are not represented.
Fig 4.
Residues substituted in variants conferring discriminative activity.
A) Library Random 2. B: Libraries Random Tot and Random Rec, combined. Green: clear discrimination for hydrolysis of short-chain triglycerides (tributyrin). Magenta: clear discrimination for hydrolysis of the long-chain triglycerides (olive oil). Purple: residues substituted in distinct variants showing opposite discriminative phenotypes. A PEG-4 molecule is shown in black spheres, crystallized inside the putative tunnel [22, 29].
Fig 5.
Hydrolytic activity of Tyr93 library variants with p-NO2-phenyl fatty acids.
Assays were performed in triplicate with clarified E. coli lysates. Activity is reported relative to WT Cal-A with pNO2-phenyl-palmitate. Its specific activity (S.A) = 0.4 U/mg, is set as 100%.
Fig 6.
Hydrolytic activity of discriminative variants from randomized libraries with p-NO2-phenyl fatty acids.
Assays were performed in triplicate with clarified E. coli lysates. Activity is reported relative to WT Cal-A with pNO2-phenyl-butyrate. Its specific activity (S.A.) = 0.4 U/mg, is set as 100%. Substitutions included in each variant are inset and recurring mutations are highlighted. Variant number is shown as per S1–S3 Tables.
Fig 7.
Hydrolytic activity of Gly237 variants with p-NO2-phenyl fatty acids.
Assays were performed in triplicate with clarified E. coli lysates. Activity is reported relative to WT Cal-A with pNO2-phenyl-palmitate. Its specific activity (S.A) = 0.4 U/mg, is set as 100%.