Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.
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.
From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Scott C
SC
Farrow
Peter J
PJ
Facchini
D044182:Benzylisoquinolines / Q000737:chemistry / Q000378:metabolism*; D001600:Berberine Alkaloids / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002853:Chromatography, Liquid; D049308:Dioxygenases / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D020868:Gene Silencing; D007700:Kinetics; D013058:Mass Spectrometry; D008745:Methylation; D009892:Opium / Q000378:metabolism*; D010207:Papaver / Q000201:enzymology*; D010802:Phylogeny; D013379:Substrate Specificity; D014780:Viruses
D044182:Benzylisoquinolines; D001600:Berberine Alkaloids; D005557:Formaldehyde; D009892:Opium; D049308:Dioxygenases; C109505:allocryptopine
Dioxygenase; Gene Silencing; Mass Spectrometry (MS); Metabolic Regulation; Plant Biochemistry; Plant Physiology; Secondary Metabolism