Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

  • FEBS Lett. 2014 Jan 3;588(1):198-204. doi: 10.1016/j.febslet.2013.11.037.
Thu-Thuy T Dang  1 ,  Peter J Facchini  2
Affiliations
  • 1. Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
  • 2. Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address: [email protected].
PMID: 24316226 DOI: 10.1016/j.febslet.2013.11.037
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.

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.