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Báo cáo khoa học: Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica

(S)-Stylopine is an important intermediate in the biosynthesis of benzophe-nanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine bio-synthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, | ỊFEBS Journal Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica Nobuhiro Ikezawa1 Kinuko Iwasa2 and Fumihiko Sato1 1 Division of Integrated Life Science Graduate Schoolof Biostudies Kyoto University Japan 2 Kobe PharmaceuticalUniversity Japan Keywords alkaloid biosynthesis cytochrome P450 Eschscholzia californica methylenedioxy bridge-forming enzyme stylopine synthase Correspondence F. Sato Division of Integrated Life Science Graduate School of Biostudies Kyoto University Kyoto 606-8502 Japan Fax 81 75 753 6398 Tel. 81 75 753 6381 E-mail fsato@lif.kyoto-u.ac.jp Note The nucleotide sequences reported in this paper have been submitted to the DDBJ GenBank EMBL Data Bank under the accession numbers AB126257 CYP719A2 and AB126256 CYP719A3 Received 6 September 2006 revised 23 November 2006 accepted 15 December 2006 doi 10.1111 j.1742-4658.2007.05652.x S -Stylopine is an important intermediate in the biosynthesis of benzophe-nanthridine alkaloids such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 CYP719 involved in berberine biosynthesis has been cloned from Coptis japonica Ikezawa N Tanaka M Nagayoshi M Shinkyo R Sakaki T Inouye K Sato F 2003 J Biol Chem 278 38557-38565 no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Esch-scholzia californica cells. Heterologous expression in Saccharomyces cerevisi-ae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and .