Epoxide Stereochemistry Controls Regioselective Ketoreduction in Epoxyquinoid Biosynthesis

Area of Science:

• Natural Product Biosynthesis
• Enzymology
• Organic Chemistry

Background:

• Epoxyquinoids are structurally diverse natural products with potential in drug discovery.
• Understanding their biosynthetic pathways is crucial for chemical biology applications.
• The biosynthesis of epoxyquinoids, including (-)-asperpentyn, remains largely uncharacterized.

Purpose of the Study:

• To fully elucidate the biosynthetic pathway of (-)-asperpentyn.
• To identify and characterize the key enzymes involved in its formation.
• To understand the mechanisms of stereochemical control during the biosynthesis.

Main Methods:

• Heterologous reconstitution in *Aspergillus oryzae*
• Chemical synthesis and chiral analysis
• Biochemical assays and enzyme kinetics
• Site-directed mutagenesis

Main Results:

• Identified FAD-binding monooxygenase AtyG, cupin domain protein AtyE, and ketoreductases AtyD and AtyC as critical enzymes.
• AtyE performs stereoselective epoxidation; AtyD and AtyC control stereochemistry in ketoreductions.
• Enzyme regioselectivity, particularly AtyD's, is influenced by substrate configuration.
• Generated four new epoxyquinols and four new epoxyhydroquinones, providing mechanistic insights.
• Demonstrated successful in vitro conversion of a precursor to (+)-asperpentyn using engineered enzymes.

Conclusions:

• The study comprehensively details the stereocontrolled biosynthesis of (-)-asperpentyn.
• Enzymatic insights gained expand the toolkit for generating complex epoxyquinoid natural products.
• The identified enzymes and pathways offer new avenues for synthetic biology and drug discovery.