Rational design of a cyclohexanone dehydrogenase for enhanced α,β-desaturation and substrate specificity

Area of Science:

• Biocatalysis and Green Chemistry
• Enzyme Engineering
• Structural Biology

Background:

• Selective α,β-desaturation of cyclic carbonyl compounds is vital for synthesizing steroids and bioactive molecules.
• Current methods often lack the green chemistry principles desired for sustainable synthesis.
• Cyclic carbonyl compounds are core structures in many important natural and synthetic molecules.

Purpose of the Study:

• To elucidate the structure and mechanism of a novel cyclohexanone dehydrogenase (CHD) from Alicycliphilus denitrificans.
• To engineer the CHD enzyme for enhanced activity, substrate scope, and application in green synthesis.
• To provide a foundation for rational enzyme design for regioselective α,β-desaturation.

Main Methods:

• X-ray crystallography to determine the de novo structure of CHD and its complex with cyclohexanone.
• Enzyme assays to investigate substrate specificity against various cyclic ketones, lactones, and lactams.
• Molecular dynamic simulations to guide protein engineering efforts for improved enzyme functionality.

Main Results:

• The de novo structure of Alicycliphilus denitrificans CHD was solved, revealing active site interactions and cofactor proximity.
• A Y195F variant provided insights into substrate binding and mechanistic roles of active site residues.
• Engineered W113A variant exhibited improved activity and accepted bulkier substrates like dihydrocoumarin due to an altered active site.

Conclusions:

• The engineered CHD enzyme offers a promising biocatalytic tool for regioselective α,β-desaturation.
• Structural and mechanistic insights facilitate rational design of enzymes for green synthesis.
• This work enables the bespoke synthesis of valuable bioactive molecules through enzyme engineering.