title: Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.
abstract: Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction.
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Takemura
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Ikezawa
authors.forename: Kinuko
authors.initials: K
authors.lastname: Iwasa
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D053119:Benzophenanthridines / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D031675:Eschscholzia / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D007546:Isoquinolines / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D053119:Benzophenanthridines; D007546:Isoquinolines; D003577:Cytochrome P-450 Enzyme System; C005705:sanguinarine