Poster: Secondary Metabolism
Abs #
281: Molecular cloning and characterization of CYP719, a methylenedioxy bridge forming enzyme belonging to a novel P450 family, from cultured Coptis japonica cells
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Presenter: |
Ikezawa, Nobuhiro , ikezawa@kais.kyoto-u.ac.jp |
Authors | Ikezawa, Nobuhiro (A) Tanaka, Masaru (B) Shinkyo, Raku (B) Sakaki, Toshiyuki (B) Inouye, Kuniyo (B) Sato, Fumihiko (A) (B) | | Affiliations: |
(A): Graduate School of Biostudies, Kyoto University
(B): Graduate School of Agriculture, Kyoto University
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Of thirteen steps in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, two are catalyzed by cytochromes P450 (P450). Interestingly, one of them catalyzes a cyclization of an ortho-methoxyphenol, which is unique and a difficult reaction to mimic in organic chemistry. We report here the isolation of this novel methylenedioxy bridge forming P450 cDNA from cultured Coptis japonica cells.
Two P450 cDNAs were isolated from a cDNA library of high berberine-producing Coptis japonica cells, using PCR fragments amplified with primers designed for the conserved region of P450. Sequence analysis showed that one Coptis japonica P450 belonged to a novel P450 family (designated CYP719). Further, heterologous expression in yeast confirmed that it had the activity of methylenedioxy bridge forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). Another P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3f-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3f-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)-Scoulerine was not the substrate of CYP719, indicating that other P450, i.e., (S)-cheilanthifoline synthase is needed in (S)-stylopine biosynthesis. All berberine biosynthetic genes including CYP719 and CYP80B2 were highly expressed in high berberine-producing cultured Coptis japonica cells and moderately in root, suggesting the coordinated regulation of gene expression of biosynthetic genes.
