Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot

Abstract

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217-245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

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].

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%.