title: Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.
abstract: Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
authors: Guillaume A W
authors: GA
authors: Beaudoin
authors: Peter J
authors: PJ
authors: Facchini
mesh_terms: D053119:Benzophenanthridines / Q000096:biosynthesis*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D007546:Isoquinolines; D009892:Opium; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
chemical_list: D053119:Benzophenanthridines; D018076:DNA, Complementary; D007546:Isoquinolines; D010940:Plant Proteins; D011994:Recombinant Proteins; D009892:Opium; D003577:Cytochrome P-450 Enzyme System; C005705:sanguinarine; C583049:(S)-cis-N-methylstylopine 14-hydroxylase, Papaver somniferum