Redirecting a Native Ene-Reductase Toward Desaturation With Reverse Enantioselectivity.

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.