Mechanistic Insights into the CC Desaturation Catalyzed by an Fe(II) and αKG‐Dependent Oxygenase AndA

Fungal meroterpenoids, bioactive natural products with complex molecular frameworks, acquire their structural diversity partially through nonheme Fe(II)/α-ketoglutarate(αKG)-dependent enzymes. These enzymes use a high-valent Fe(IV)-oxo intermediate to drive diverse oxidative transformations. AndA, an Fe(II)/αKG oxygenase pivotal to anditomin biosynthesis, catalyzes regioselective C1C2 desaturation followed by skeletal rearrangement. While the isomerization step has been characterized, the mechanistic basis for desaturation over competing hydroxylation pathways remains enigmatic. Herein, molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations are employed to unravel how AndA avoids hydroxylation to achieve regioselective desaturation. The findings reveal the Fe(IV)-oxo intermediate, adopting two distinct coordination modes, a pentacoordinate (5C) and hexacoordinate (6C) geometry, differentiated by succinate coordination. The more reactive 5C species selectively abstracts the C2H hydrogen, initiating desaturation. Crucially, CO₂ generated in situ from αKG decarboxylation reacts with the resultant Fe(III)-OH complex, forming an Fe(III)-bicarbonate complex. This species sterically and electronically blocks OH rebound to the substrate. The Fe(III)-bicarbonate then abstracts a C1 hydrogen atom, completing the formation of the C1C2 double bond. These insights resolve the mechanism of AndA-catalyzed regioselective desaturation and demonstrate how CO₂-mediated coordination modulates oxidative fate, advancing mechanistic understanding of product control in this enzyme class.