Designing enzymes with complementary enantioselectivity profiles

Scala’s underlying active-site design technology generates enantiodivergent, stable UPO variants

Why it matters

Enantioselectivity, the preference for one mirror-image product over another, is crucial in pharmaceuticals and fine chemicals, where the two forms often have very different effects. Yet controlling enantioselectivity is notoriously difficult. Scala’s underlying active-site design technology was applied to an unspecific peroxygenase (UPO), generating a stable and diverse repertoire of variants with dramatically altered selectivity profiles.

Problem

Unspecific peroxygenases (UPOs) can carry out highly valuable reactions by inserting oxygen into strong C–H bonds, making them attractive for drug and chemical synthesis. Their challenge is enantioselectivity: because the access channel to their active site is large and flexible, it is very challenging to control whether the enzyme produces the left- or right-handed form of a molecule. This limits their usefulness in applications where the exact product form is critical.

What was done

Starting from PaDa-I, a laboratory-evolved UPO with good stability and expression, Scala’s underlying active-site design technology was applied to propose new variants. The method introduced combinations of 4–5 mutations in the enzyme’s active site and selected the most promising designs for testing. In a single round, 30 variants were created, of which 24 were stable, well-expressed, and functional.

Results

• Design scope: Thirty UPO variants were generated, each carrying 4–5 active-site mutations;

• Expression: 24/30 were expressed as stable, active enzymes.

• Enantioselectivity: Across four substrates, 12 variants improved enantiomeric excess (ee) >20 %, and 5 out of the 12 variants inverted preference relative to wild type.

• Catalytic efficiency: For ethylbenzene oxidation, variants d2 and d4 showed 4× and 10× higher kcat/Km, respectively, while variant d28 improved ~2× and inverted product configuration.

• Stability: All active variants retained or increased thermostability, with no activity–stability trade-off observed.

Impact

Applying Scala’s underlying active-site design technology generated a diverse panel of UPO variants with complementary enantioselectivity in a single design round. Instead of relying on long, iterative mutagenesis campaigns, Scala's method delivered ready-made enzyme variants that are both efficient and enantiodivergent. This provides a practical starting point for selective oxyfunctionalization in pharmaceuticals and fine chemicals, expanding the range of molecules that can be accessed through biocatalysis.

Data highlights

• Enantiodivergence: across all tested substrates, designs either improved enantioselectivity or switched preference to the opposite enantiomer (shown in the heat map, where several designs display the reverse profile compared to PaDa-I)

  
  

  

  
  

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• Catalytic efficiency (ethylbenzene): Catalytic efficiency (kcat/Km) combines how fast an enzyme works with how well it binds its substrate; higher values mean more efficient catalysis. Variants d2 and d4 showed 4× and 10× higher kcat/Km, respectively, while variant d28 improved ~2× and inverted product configuration.

  
  

  

  
  

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References

Gómez de Santos P, Mateljak I, Hoang MD, Fleishman SJ, Hollmann F, Alcalde M. Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C–H Oxyfunctionalization Reactions. J. Am. Chem. Soc. 2023;145:3443–3453. doi:10.1021/jacs.2c11118.