noscapine and canadine

We have characterized four sequential enzymes that transform 1-hydroxy-N-methylcanadine to narcotoline hemiacetal, completing our elucidation of noscapine biosynthesis in opium poppy. Two cytochromes P450 catalyze hydroxylations at C13 and C8 on the protoberberine scaffold, the latter step inducing ring opening and the formation of an aldehyde moiety. Acetylation at C13 before C8 hydroxylation introduces a protective group subsequently hydrolyzed by a carboxylesterase, which triggers rearrangement to a cyclic hemiacetal.

Topics: Acetylation; Berberine; Biosynthetic Pathways; Cyclization; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Silencing; Hydroxylation; Noscapine; Papaver; Plant Proteins; Recombinant Proteins; Substrate Specificity

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.

Topics: Amino Acid Sequence; Berberine; Berberine Alkaloids; Biosynthetic Pathways; Chromatography, Liquid; Cloning, Molecular; Cytochrome P-450 Enzyme System; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Silencing; Immunoblotting; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Noscapine; Papaver; Phylogeny; Plant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Substrate Specificity