Abstract
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.
Highlights
Thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM) catalyze late steps of morphine biosynthesis in opium poppy
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
O-demethylation of allocryptopine by T6ODM was characterized by a reaction product with a quasi-molecular ion ([M ϩ H]ϩ) reduced by 14 atomic mass units compared with the substrate (Fig. 5, A and B), whereas O,O-demethylenation by PODA and CODM resulted in a quasi-molecular ion reduced by 12 atomic mass units (Fig. 5, A, C, and D)
Summary
Thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM) catalyze late steps of morphine biosynthesis in opium poppy. 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. The discovery of T6ODM and CODM provided a rational basis for hypothesizing the occurrence of additional O-demethylation events in BIA metabolism and prompted a reevaluation of established biosynthetic pathways [5] Such consideration revealed several alkaloid end products with functional group substitution patterns that suggested the O-demethylation of upstream pathway intermediates. Several substrates undergo regiospecific O-demethylation similar to the late steps in morphine biosynthesis, CODM and PODA catalyze the O,O-demethylenation of methylenedioxy bridges on protopine alkaloids and, in the case of PODA, on protoberberine alkaloids containing this functional group. We extend the function of ODDs to include O,O-demethylenation and provide evidence for an expanded role of ODDs in BIA metabolism
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