Unraveling the Mechanism of the Oxidative C-C Bond Coupling Reaction Catalyzed by Deoxypodophyllotoxin Synthase

Deoxypodophyllotoxin synthase (DPS), a nonheme Fe(II)/2-oxoglutarate (2OG)-dependent oxygenase, is a key enzyme that is involved in the construction of the fused-ring system in (-)-podophyllotoxin biosynthesis by catalyzing the C-C coupling reaction. However, the mechanistic details of DPS-catalyzed ring formation remain unclear. Herein, our quantum mechanics/molecular mechanics (QM/MM) calculations reveal a novel mechanism that involves the recycling of CO₂ (a product of decarboxylation of 2OG) to prevent the formation of hydroxylated byproducts. Our results show that CO₂ can react with the Fe^III-OH species to generate an unusual Fe^III-bicarbonate species. In this way, hydroxylation is avoided by consuming the OH group. Then, the C-C coupling followed by desaturation yields the final product, deoxypodophyllotoxin. This work highlights the crucial role of the CO₂ molecule, generated in the crevice between the iron active site and the substrate, in controlling the reaction selectivity.