title: Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana.
abstract: Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (S)-cheilanthifoline from (S)-scoulerine and of (S)-stylopine from (S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (S)-scoulerine to (S)-cheilanthifoline (K(m) 1.9±0.3; k(cat)/K(m) 1.7), CYP719A13 converted (S)-tetrahydrocolumbamine to (S)-canadine (K(m) 2.7±1.3; k(cat)/K(m) 12.8), (S)-cheilanthifoline to (S)-stylopine (K(m) 5.2±3.0; k(cat)/K(m) 2.6) and (S)-scoulerine to (S)-nandinine (K(m) 8.1±1.9; k(cat)/K(m) 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana.
authors.affiliation: Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA. chavez_luisa@yahoo.com.mx
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Díaz Chávez
authors.forename: Megan
authors.initials: M
authors.lastname: Rolf
authors.forename: Andreas
authors.initials: A
authors.lastname: Gesell
authors.forename: Toni M
authors.initials: TM
authors.lastname: Kutchan
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D031674:Argemone / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053119:Benzophenanthridines / Q000378:metabolism*; D044182:Benzylisoquinolines / Q000378:metabolism; D001600:Berberine Alkaloids / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D007546:Isoquinolines / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D000900:Anti-Bacterial Agents; D053119:Benzophenanthridines; D044182:Benzylisoquinolines; D001600:Berberine Alkaloids; D007546:Isoquinolines; D003577:Cytochrome P-450 Enzyme System; C005705:sanguinarine; C014212:stylopine; C003298:reticuline