Dioxygenases Catalyze O-Demethylation and O,O-Demethylenation with Widespread Roles in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

Background: Thebaine 6- O -demethylase (T6ODM) and codeine O -demethylase (CODM) catalyze late steps of morphine biosynthesis in opium poppy. Results: New dealkylation reactions, including O,O -demethylenation, have been detected for T6ODM, CODM, and other 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODDs). Virus-induced gene silencing supports physiological functions. Conclusion: Certain ODDs are multifunctional dealkylating enzymes with widespread roles in benzylisoquinoline alkaloid metabolism. Significance: Enzymes responsible for O -demethylation and O,O -demethylenation in plant alkaloid biosynthesis have been discovered. In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6- O- demethylase (T6ODM) and codeine O- demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[ c ]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O -dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O -demethylation and O,O -demethylenation. Enzymes catalyzing O,O -demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O -demethylenation by CODM and protopine O -dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[ c ]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[ c ]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.