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J. Biol. Chem., Vol. 283, Issue 14, 8810-8821, April 4, 2008

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Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C–C Phenol Coupling of (S)-Reticuline in Magnoflorine Biosynthesis, from Cultured Coptis japonica Cells^*

Nobuhiro Ikezawa, Kinuko Iwasa, and Fumihiko Sato¹

From the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502 and the Kobe Pharmaceutical University, Kobe, Hyogo 658-8558, Japan

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C–C phenol-coupling and intermolecular C–O phenol-coupling reactions. We report here the isolation and characterization of a C–C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C–O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C–C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C–C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.

Received for publication, June 20, 2007 , and in revised form, January 15, 2008.

^* This research was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to F. S.) and by a fellowship from the Japan Society for the Promotion of Science (to N. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB288053.

¹ To whom correspondence should be addressed. Tel.: 81-75-753-6381; Fax: 81-75-753-6398; E-mail: fsato{at}lif.kyoto-u.ac.jp.

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