Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy |
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| Authors: | Thu-Thuy T. Dang Peter J. Facchini |
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| Affiliation: | Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada |
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| 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. |
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| Keywords: | BIA, benzylisoquinoline alkaloid CID, collision-induced dissociation CPR, cytochrome P450 reductase CYP, cytochrome P450 ESI, electrospray ionization GAPDH, glyceraldehyde 3-phosphate dehydrogenase LC&ndash MS/MS, liquid chromatography&ndash tandem mass spectrometry SOMT, scoulerine 9-O-methyltransferase TNMT, tetrahydroprotoberberine N-methyltransferase TRV, tobacco rattle virus VIGS, virus-induced gene silencing |
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