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Updated: Jun 13, 2026
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Redirecting a Native Ene-Reductase Toward Desaturation With Reverse Enantioselectivity
Qing-Qing Zeng1,2, Cristina Berga3, Carla Calvó-Tusell3,4
1Academy For Advanced Interdisciplinary Studies, Peking University, Beijing, China.
Angewandte Chemie (International Ed. in English)
|June 12, 2026
Summary
Researchers engineered an old yellow enzyme (OYE) for chiral enone synthesis, achieving high enantioselectivity and yields. This biocatalytic system provides a stereocomplementary approach to valuable synthetic intermediates.
Area of Science:
- Biocatalysis and synthetic organic chemistry
- Enzyme engineering and directed evolution
Background:
- Chiral enones are crucial building blocks in pharmaceuticals and natural products.
- Existing enzymatic desaturation methods for cyclohexenones lack stereocomplementary options.
Purpose of the Study:
- To develop a novel biocatalytic system for stereoselective synthesis of chiral enones.
- To engineer an old yellow enzyme (OYE) for desaturation activity with complementary stereoselectivity.
Main Methods:
- Directed evolution of XenA, an old yellow enzyme from Pseudomonas putida.
- Protein engineering to redirect catalytic function from reduction to desaturation.
- Biocatalytic screening and characterization of enzyme variants.
Main Results:
- Engineered XenA variant (XenA_4) with 46 mutations demonstrated efficient desaturation of cyclohexanones.
- Achieved high enantioselectivity (85%-99% ee) and yields (32%-98%).
- The engineered enzyme exhibits increased thermal stability (11°C higher melting temperature).
Conclusions:
- Protein engineering successfully repurposed XenA for stereoselective enone synthesis.
- The dimeric structure of XenA plays a key role in controlling stereoselectivity.
- This provides a valuable stereocomplementary biocatalytic tool for chiral enone production.
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