Bienzyme-Catalytic and Dioxygenation-Mediated Anthraquinone Ring Opening

Area of Science:

• Biochemistry
• Molecular Biology
• Mycology

Background:

• Anthraquinone ring cleavage is vital for fungal seco-anthraquinone biosynthesis.
• The exact mechanism, particularly the C-10-C-4a bond cleavage, has remained unclear, with Baeyer-Villiger oxidation previously proposed.
• Genetic, enzymatic, and chemical data on this ring-opening process were lacking.

Purpose of the Study:

• To elucidate the revised mechanism of anthraquinone ring opening in questin biosynthesis.
• To identify the specific enzymes and cofactors involved in the C-10-C-4a bond cleavage.
• To demonstrate the universality of the proposed ring-opening pathway.

Main Methods:

• *In vivo* gene disruption studies to identify key genes.
• *In vitro* enzymatic assays to characterize enzyme function.
• ¹⁸O chasing experiments to trace oxygen incorporation during the reaction.

Main Results:

• The reductase GedF, utilizing NADPH, was confirmed to reduce the C-10 keto group of questin to a hydroxyl group.
• The atypical cofactor-free dioxygenase GedK was identified as the enzyme responsible for cleaving the C-10-C-4a bond of the resulting questin hydroquinone.
• This enzymatic action yields desmethylsulochrin, revealing a novel ring-opening mechanism.

Conclusions:

• A revised, bienzyme-catalytic mechanism for anthraquinone ring opening has been elucidated.
• The pathway involves sequential reduction by GedF and cofactor-free dioxygenation by GedK.
• This mechanism is proposed to be a universal pathway in fungal seco-anthraquinone biosynthesis.