title: Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy.
abstract: Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (Km=4.63±0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors: Thu-Thuy T
authors: TT
authors: Dang
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address: pfacchin@ucalgary.ca.
authors: Peter J
authors: PJ
authors: Facchini
mesh_terms: D000595:Amino Acid Sequence; D001599:Berberine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001600:Berberine Alkaloids / Q000737:chemistry / Q000378:metabolism; D053898:Biosynthetic Pathways; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009665:Noscapine / Q000737:chemistry / Q000378:metabolism*; D010207:Papaver / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001600:Berberine Alkaloids; C586635:CYP719A21 protein, Papaver; D010940:Plant Proteins; D001599:Berberine; D009665:Noscapine; D003577:Cytochrome P-450 Enzyme System; C092892:tetrahydrocolumbamine; C004645:canadine
keywords: BIA; Benzylisoquinoline alkaloid; CID; CPR; CYP; Cytochrome P450; ESI; GAPDH; LC–MS/MS; Methylenedioxy bridge; Noscapine biosynthesis; Papaver somniferum; SOMT; TNMT; TRV; VIGS; Virus-induced gene silencing; benzylisoquinoline alkaloid; collision-induced dissociation; cytochrome P450; cytochrome P450 reductase; electrospray ionization; glyceraldehyde 3-phosphate dehydrogenase; liquid chromatography–tandem mass spectrometry; scoulerine 9-O-methyltransferase; tetrahydroprotoberberine N-methyltransferase; tobacco rattle virus; virus-induced gene silencing