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

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. 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. Isoquinoline alkaloids are a large group of alkaloids and include many pharmacologically useful compounds; e.g. the analgesic morphinan alkaloid morphine, the anti-tussive alkaloid codeine, and the anti-microbial alkaloids berberine and sanguinarine. Due to the importance of these pharmaceutically useful alkaloids, their biosynthetic pathways have been well investigated, and several of them have been completely clarified at the enzyme level (1Croteau R. Kutchan T.M. Lewis N. Buchanan B. Gruissem W. Jones R. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD2000: 1250-1318Google Scholar, 2Zenk M.H. Pure & Appl. Chem. 1994; 66: 2023-2028Crossref Scopus (54) Google Scholar, 3Kutchan T.M. Cordell G.A. The Alkaloids–Chemistry and Biology, vol. 50. Academic Press, San Diego, CA1998: 257-316Google Scholar). It is now known that many isoquinoline alkaloids share a common biosynthetic pathway from l-tyrosine to the key intermediate (S)-reticuline. (S)-Reticuline is a central precursor of various types of isoquinoline alkaloids such as morphinans, aporphines, pavines, protoberberines, protopines, and benzophenanthridines (1Croteau R. Kutchan T.M. Lewis N. Buchanan B. Gruissem W. Jones R. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD2000: 1250-1318Google Scholar, 4Preininger V. Brossi A. The Alkaloids–Chemistry and Pharmacology, vol. 29. Academic Press, San Diego, CA1986: 1-98Google Scholar). Although the molecular origin of this chemical diversity has not yet been clarified, recent studies have shown that many of their oxidative steps are catalyzed by cytochrome P450s (P450) 2The abbreviations used are: P450cytochrome P450ESTexpressed sequence tagRACErapid amplification of cDNA endsHPLChigh performance liquid chromatographyLC-MSliquid chromatography-mass spectroscopyCNMTS-adenosyl-l-methionine:coclaurine N-methyltransferaseCjCNMTCNMT of C. japonicaBBEberberine bridge enzymeMS/MStandem mass spectrometry. (Fig. 1) (1Croteau R. Kutchan T.M. Lewis N. Buchanan B. Gruissem W. Jones R. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD2000: 1250-1318Google Scholar, 2Zenk M.H. Pure & Appl. Chem. 1994; 66: 2023-2028Crossref Scopus (54) Google Scholar, 3Kutchan T.M. Cordell G.A. The Alkaloids–Chemistry and Biology, vol. 50. Academic Press, San Diego, CA1998: 257-316Google Scholar, 5Pauli H.H. Kutchan T.M. Plant J. 1998; 13: 793-801Crossref PubMed Scopus (183) Google Scholar, 6Ikezawa N. Tanaka M. Nagayoshi M. Shinkyo R. Sakaki T. Inouye K. Sato F. J. Biol. Chem. 2003; 278: 38557-38565Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 7Bauer W. Zenk M.H. Phytochemistry. 1991; 30: 2953-2961Crossref Scopus (80) Google Scholar, 8Ikezawa N. Iwasa K. Sato F. FEBS J. 2007; 274: 1019-1035Crossref PubMed Scopus (99) Google Scholar, 9Gerardy R. Zenk M.H. Phytochemistry. 1993; 32: 79-86Crossref Scopus (130) Google Scholar, 10Stadler R. Zenk M.H. J. Biol. Chem. 1993; 268: 823-831Abstract Full Text PDF PubMed Google Scholar, 11Kraus P.F.X. Kutchan T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2071-2075Crossref PubMed Scopus (155) Google Scholar). Members of the P450 family are found in a very large number of species, especially in the plant kingdom (246 and 356 species in Arabidopsis thaliana and Oryza sativa in contrast to 57 and 84 species in human and Drosophila melanogaster) (12Nelson D.R. Schuler M.A. Paquette S.M. Werck-Reichhart D. Bak S. Plant Physiol. 2004; 135: 756-772Crossref PubMed Scopus (357) Google Scholar, 13Nelson D.R. Zeldin D.C. Hoffman S.M.G. Maltais L.J. Wain H.M. Nebert D.W. Pharmacogenetics. 2004; 14: 1-18Crossref PubMed Scopus (754) Google Scholar), and many of them have been shown to be involved in plant secondary metabolism (14Chapple C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 311-343Crossref PubMed Scopus (356) Google Scholar, 15Werck-Reichhart D. Bak S. Paquette S. Somerville C.R. Meyerowitz E.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2002Google Scholar). cytochrome P450 expressed sequence tag rapid amplification of cDNA ends high performance liquid chromatography liquid chromatography-mass spectroscopy S-adenosyl-l-methionine:coclaurine N-methyltransferase CNMT of C. japonica berberine bridge enzyme tandem mass spectrometry. In the biosyntheses of isoquinoline alkaloids, P450-mediated hydroxylation, methylenedioxy bridge formation, and phenol-coupling reactions have been reported. Although members of the CYP80B subfamily catalyze hydroxylation from (S)-N-methylcoclaurine to (S)-3′-hydroxy-N-methylcoclaurine in (S)-reticuline biosynthesis (5Pauli H.H. Kutchan T.M. Plant J. 1998; 13: 793-801Crossref PubMed Scopus (183) Google Scholar), other P450 reactions, including methylenedioxy bridge formation and phenol-coupling reactions, are involved in the biosynthesis of rather specific isoquinoline alkaloids (6Ikezawa N. Tanaka M. Nagayoshi M. Shinkyo R. Sakaki T. Inouye K. Sato F. J. Biol. Chem. 2003; 278: 38557-38565Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 7Bauer W. Zenk M.H. Phytochemistry. 1991; 30: 2953-2961Crossref Scopus (80) Google Scholar, 8Ikezawa N. Iwasa K. Sato F. FEBS J. 2007; 274: 1019-1035Crossref PubMed Scopus (99) Google Scholar, 9Gerardy R. Zenk M.H. Phytochemistry. 1993; 32: 79-86Crossref Scopus (130) Google Scholar, 10Stadler R. Zenk M.H. J. Biol. Chem. 1993; 268: 823-831Abstract Full Text PDF PubMed Google Scholar, 11Kraus P.F.X. Kutchan T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2071-2075Crossref PubMed Scopus (155) Google Scholar). In the biosynthesis of berberine (protoberberine type) and macarpine (benzophenanthridine type), three methylenedioxy bridge-forming reactions, i.e. canadine synthase, cheilanthifoline synthase, and stylopine synthase reactions, have been reported (3Kutchan T.M. Cordell G.A. The Alkaloids–Chemistry and Biology, vol. 50. Academic Press, San Diego, CA1998: 257-316Google Scholar). Methylenedioxy bridge formation is the cyclization of an ortho-methoxyphenol moiety, and is commonly found in many secondary metabolites, including lignans. Our recent research identified canadine synthase cDNA from Coptis japonica (CYP719A1) (6Ikezawa N. Tanaka M. Nagayoshi M. Shinkyo R. Sakaki T. Inouye K. Sato F. J. Biol. Chem. 2003; 278: 38557-38565Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and stylopine synthase cDNAs from Eschscholzia californica (CYP719A2 and CYP719A3) (8Ikezawa N. Iwasa K. Sato F. FEBS J. 2007; 274: 1019-1035Crossref PubMed Scopus (99) Google Scholar). Currently, P450 species that catalyze methylenedioxy bridge-forming reactions are rather rare: only the CYP719A subfamily has been found in isoquinoline alkaloid biosynthesis and the CYP81Q subfamily has been found in sesamin biosynthesis (16Ono E. Nakai M. Fukui Y. Tomimori N. Fukuchi-Mizutani M. Saito M. Satake H. Tanaka T. Katsuta M. Umezawa T. Tanaka Y. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10116-10121Crossref PubMed Scopus (117) Google Scholar). Two types of P450-mediated phenol-coupling reactions have been reported in isoquinoline alkaloid biosynthesis. One is the intramolecular C–C phenol-coupling reaction, catalyzed by salutaridine synthase, from (R)-reticuline to produce salutaridine in morphine biosynthesis (9Gerardy R. Zenk M.H. Phytochemistry. 1993; 32: 79-86Crossref Scopus (130) Google Scholar). The other is the intermolecular C–O phenol-coupling reaction, which is catalyzed by berbamunine synthase to make berbamunine (bisbenzylisoquinoline alkaloid) using (R)- and (S)-N-methylcoclaurine as substrates (10Stadler R. Zenk M.H. J. Biol. Chem. 1993; 268: 823-831Abstract Full Text PDF PubMed Google Scholar, 11Kraus P.F.X. Kutchan T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2071-2075Crossref PubMed Scopus (155) Google Scholar). So far, only berbamunine synthase has been cloned from Berberis stolonifera and has been designated CYP80A1 (11Kraus P.F.X. Kutchan T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2071-2075Crossref PubMed Scopus (155) Google Scholar). Japanese goldthread C. japonica (Ranunculaceae) produces a large amount of berberine and moderate amounts of several other isoquinoline alkaloids (Fig. 2). Although our previous study showed that two P450 genes (CYP80B2 and CYP719A1) were involved in berberine biosynthesis (6Ikezawa N. Tanaka M. Nagayoshi M. Shinkyo R. Sakaki T. Inouye K. Sato F. J. Biol. Chem. 2003; 278: 38557-38565Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), there has been no other previous identification of P450 genes involved in isoquinoline alkaloid biosynthesis in C. japonica. Because the biosyntheses of magnoflorine and coptisine were suggested, based on their chemical structures, to require oxidative steps from their respective precursors, (S)-reticuline and (S)-scoulerine, we speculated that they include several P450 reactions. Based on this idea, to clone their biosynthetic genes, we decided to search for candidate P450 genes using 4032 expressed sequence tags (ESTs) prepared from cultured C. japonica cells. As a result, we found a novel P450 cDNA fragment, which was significantly similar to B. stolonifera CYP80A1 and isolated its full-length cDNA. This novel P450, designated CYP80G2 by the P450 nomenclature committee, was heterologously expressed in yeast to characterize its enzyme activity. Recombinant CYP80G2 showed intramolecular C–C phenol-coupling activity to convert (S)-reticuline to corytuberine. This is the first report of the isolation of cDNA of a eukaryotic microsomal-bound P450, which catalyzes a C–C phenol-coupling reaction. A detailed substrate specificity analysis of CYP80G2 was conducted to obtain information about its reaction mechanism and substrate recognition. In addition, we discuss the role of the unique amino acid residue in the helix I region of CYP80G2 in its C–C phenol-coupling reaction. We also discuss the involvement of CYP80G2 in magnoflorine biosynthesis, because corytuberine has been proposed to be the precursor of magnoflorine (17Bhakuni D.S. Jain S. Singh R.S. Tetrahedron. 1980; 36: 2525-2528Crossref Scopus (10) Google Scholar, 18Milanowski D.J. Winter R.E.K. Elvin-Lewis M.P.F. Lewis W.H. J. Nat. Prod. 2002; 65: 814-819Crossref PubMed Scopus (43) Google Scholar), an aporphine-type alkaloid produced by cultured C. japonica cells. Plant Material—The original cultured cells were induced from rootlets of C. japonica Makino var. dissecta (Yatabe) Nakai. A cell line (156-1) that produces large amounts of alkaloids was established and subcultured as described previously (19Sato F. Yamada Y. Phytochemistry. 1984; 23: 281-285Crossref Scopus (153) Google Scholar). Ten-day-old cultured cells were harvested and used for the extraction of mRNA and alkaloids. Metabolite Analysis of Cultured C. japonica Cells—Cells were collected from a 10-day-old cell culture and extracted with MeOH, and an aliquot of the MeOH extract was analyzed directly by high performance liquid chromatography (HPLC). Reversed-phase HPLC was performed with a Shimadzu LC-10A system: column, TSKgel ODS-80TM (4.6 × 250 mm; Tosoh); solvent system, acetonitrile/H2O/acetic acid (30:69:1); flow rate, 0.8 ml/min; detection, absorbance measurement at 280 nm with an SPD6A photodiode array detector. Chemicals—(S)-Reticuline was a gift from Dr. P. J. Facchini of the University of Calgary. (S)-N-Methylcoclaurine was a gift from Dr. Y. Sugimoto of Kobe University. (S)-Coclaurine was a gift from Dr. N. Nagakura of Kobe Pharmaceutical University. Magnoflorine was a gift from Dr. R. Nishida of Kyoto University. (R,S)-Reticuline, (R,S)-norreticuline, (R,S)-6-O-methylnorlaudanosoline, and (R,S)-norlaudanosine were gifts from Mitsui Chemicals, Inc., Japan. (R,S)-Laudanosine, (R,S)-laudanosoline, and (R,S)-norlaudanosoline were purchased from Sigma-Aldrich, Inc. (R,S)-Norlaudanine and (R,S)-norpseudocodamine were prepared as described previously (20Iwasa K. Cui W. Sugiura M. Takeuchi A. Moriyasu M. Takeda K. J. Nat. Prod. 2005; 68: 992-1000Crossref PubMed Scopus (38) Google Scholar). (R,S)-Orientaline, (R,S)-codamine, (R,S)-6-O-methyllaudanosoline, (R,S)-laudanine, (R,S)-4′-O-methyllaudanosoline, and (R,S)-pseudocodamine were prepared as described previously (21Cui W. Iwasa K. Tokuda H. Kashihara A. Mitani Y. Hasegawa T. Nishiyama Y. Moriyasu M. Nishino H. Hanaoka M. Mukai C. Takeda K. Phytochemistry. 2006; 67: 70-79Crossref PubMed Scopus (35) Google Scholar). Ketoconazole was purchased from Wako Pure Chemical Industries, Ltd., Japan. Construction and Sequencing of a cDNA Library of C. japonica—A cDNA library of cultured C. japonica cells was constructed as described previously (22Choi K.B. Morishige T. Shitan N. Yazaki K. Sato F. J. Biol. Chem. 2002; 277: 830-835Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Sequencing of the cDNA library was performed for ∼4032 clones, which included redundant clones. 3Y. Kokabu, N. Kato, E. Dubouzet, J. Dubouzet, and F. Sato, unpublished data. The obtained ESTs were annotated using a BLAST search (blastx, available at www.ncbi.nlm.nih.gov/BLAST/). 5′-Rapid Amplification of cDNA Ends—5′-Rapid amplification of cDNA ends (RACE) was performed using a Gene Racer kit (Invitrogen) following the manufacturer's instructions. A total RNA sample (3 μg) from 7-day-old cultured cells was used. A gene-specific primer, 5′-GSP (5′-AAGCCATGACCGTGGGTTGAGTACC-3′), was designed. The sequence of the universal primer for 5′-RACE was given in the user manual for the kit. The resultant PCR products at ∼350 bp were subcloned into pT7Blue T-vector (Novagen), and their nucleotide sequences were determined completely. Alignment Analysis—The predicted protein sequences were aligned using ClustalW (23Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55767) Google Scholar, 24Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar) and Boxshade. Construction of Yeast Expression Vectors—The coexpression vector pGYR for P450 and yeast NADPH-P450 reductase was provided by Dr. Y. Yabusaki (Sumitomo Chemical Co., Ltd.). This vector contained glyceraldehyde-3-phosphate dehydrogenase promoter and terminator (25Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Ohkawa H. J. Biol. Chem. 1992; 267: 16497-16502Abstract Full Text PDF PubMed Google Scholar). The cloning site of pGYR was further modified to contain an SpeI site to construct pGYR-SpeI. Full-length CYP80G2 cDNA was amplified by PCR using single strand cDNAs synthesized from 1.3 μg of total RNA of cultured C. japonica cells with oligo(dT) primer and SuperScript III RNase H-reverse transcriptase (Invitrogen). The following primers were designed to introduce an SpeI site (ACTAGT, underlined): forward primer (5′-ACTAGTTTCAGAACCAAGGATAGAGATTTCAAATGG-3′) and reverse primer (5′-ACTAGTAAAACGTGAAATTTCTTATTGCCGCAAC-3′). PCR products were first subcloned into pT7Blue T-vector, and their nucleotide sequences were confirmed and then digested with SpeI to produce CYP80G2 coding fragments. The coding fragments were ligated into the SpeI site of pGYR-SpeI to generate yeast expression vector, pGS-CYP80G2. Heterologous Expression of CYP80G2 in Yeast—The expression plasmid for CYP80G2 (pGS-CYP80G2) was introduced into yeast strain AH22 (26Oeda K. Sakaki T. Ohkawa H. DNA. 1985; 4: 203-210Crossref PubMed Scopus (204) Google Scholar) by the LiCl method (27Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). These recombinant yeast cells were cultivated in concentrated SD medium (5.4 % yeast nitrogen base without amino acids, 8 % glucose, and 160 mg/l histidine) at 30 °C, 220 rpm (28Sakaki T. Shibata M. Yabusaki Y. Murakami H. Ohkawa H. DNA Cell Biol. 1990; 9: 603-614Crossref PubMed Scopus (88) Google Scholar). Yeast microsomal fractions were prepared as described previously (6Ikezawa N. Tanaka M. Nagayoshi M. Shinkyo R. Sakaki T. Inouye K. Sato F. J. Biol. Chem. 2003; 278: 38557-38565Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and suspended in a buffer (50 mm HEPES/NaOH (pH 7.6)) for the enzyme assay. Protein concentration was determined according to Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) with bovine serum albumin as a standard. Large Scale Preparation of Reaction Product of CYP80G2 and LC NMR Analysis—To determine the structure of the CYP80G2 reaction product, we converted (R,S)-reticuline to its corresponding product using CYP80G2-expressing yeast cells in vivo. CYP80G2-expressing yeast cells, grown to the logarithmic phase in concentrated SD medium at 30 °C, 220 rpm, were harvested and resuspended in 50 mm HEPES/NaOH buffer (pH 7.6) containing (R,S)-reticuline at 100 μm, and then incubated for over 30 h, which resulted in the moderate production of CYP80G2 product. The use of 50 mm HEPES/NaOH buffer (pH 7.6) was essential for this conversion, because the low pH environment, which was due to incubation in concentrated SD medium, interfered with the uptake of (R,S)-reticuline by yeast and resulted in no conversion. Because the reaction product was released into the incubation buffer, it was collected from the buffer using Sep-Pak® Plus C18 cartridges (Waters). The CYP80G2 reaction product was analyzed by LC NMR (1H NMR) as described previously (30Iwasa K. Kuribayashi A. Sugiura M. Nishiyama Y. Ichimaru M. Moriyasu M. Lee D.-U. Pharmazie. 2004; 59: 480-483PubMed Google Scholar), and its 1H NMR spectrum was compared with that of authentic corytuberine (described below). For LC NMR analysis of the CYP80G2 reaction product, a TSKgel ODS-80TM column (4.6 × 250 mm, Tosoh) was used. Preparation of Authentic Corytuberine—Authentic corytuberine was prepared by the acid-catalyzed ether cleavage of (+)-isocorydine (purchased from Sigma-Aldrich, Inc.) as follows. A solution of (+)-isocorydine in 47% HBr was refluxed for 10 min and evaporated in vacuo to give a crystalline mixture and then separated by preparative HPLC with the following system: column, COSMOSIL 5C18-AR-II (20 × 250 mm, Nacalai Tesque, Inc.); solvent system, 0.1 m NH4OAc (0.05% trifluoroacetic acid)/acetonitrile (0.05% trifluoroacetic acid). LC NMR analysis was performed with the prepared compound as described previously (30Iwasa K. Kuribayashi A. Sugiura M. Nishiyama Y. Ichimaru M. Moriyasu M. Lee D.-U. Pharmazie. 2004; 59: 480-483PubMed Google Scholar) with the following system: column, COSMOSIL 5C18-AR-II (4.6 × 150 mm; Nacalai Tesque, Inc.); solvent system, mobile phase of 0.1 m NH4OAc in D2O (0.05% trifluoroacetic acid), to which acetonitrile (0.05% trifluoroacetic acid) was added in a linear gradient from 20% to 30% at 5 min. The compound was eluted at ∼2.8 min, and corytuberine was identified based on its 1H NMR and nuclear Overhauser effect spectroscopy spectra. LC-MS/MS Analysis—LC-MS/MS analysis was performed with the CYP80G2 reaction product and authentic corytuberine, using an Applied Biosystems API3000 with the following system: column, COSMOSIL 5C18-AR-II (4.6 × 150 mm, Nacalai Tesque, Inc.); solvent system, mobile phase of 0.1 m NH4OAc (0.05% trifluoroacetic acid), to which MeOH (0.05% trifluoroacetic acid) was added in a linear gradient from 20% to 100% at 30 min. The compound was eluted at ∼5.5 min. Measurement of P450 Hemoprotein—The reduced CO-difference spectra were measured with a Shimadzu UV-3101 spectrophotometer (Kyoto, Japan) as described previously (8Ikezawa N. Iwasa K. Sato F. FEBS J. 2007; 274: 1019-1035Crossref PubMed Scopus (99) Google Scholar). The P450 hemoprotein content in the microsomal fraction of CYP80G2-expressing yeast was determined from the reduced CO-difference spectrum using a difference of 91 mm–1 ·cm–1 between the extinction coefficients at 448 and 490 nm (31Omura T. Sato R. J. Biol. Chem. 1964; 239: 2379-2385Abstract Full Text PDF PubMed Google Scholar). Assay of Enzymatic Activity—CYP80G2 activity was determined by HPLC and liquid chromatography-mass spectroscopy (LC-MS). The standard enzyme reaction mixture consisted of 50 mm HEPES/NaOH (pH 7.6), 500 μm NADPH, 50 μm substrate, and the microsomal fraction (2.2 nm P450). The assay mixture was incubated at 30 °C for 30 min, whereas 5 or 10 min of incubation was used to determine kinetic parameters or for ketoconazole inhibition and oxygen-diminishing experiments. The reaction was terminated by the addition of trichloroacetic acid at a final concentration of 2%. After protein precipitation, the amount of reaction product was determined quantitatively by reversed-phase HPLC with a Shimadzu LC-10A system: column, TSKgel ODS-80TM (4.6 × 250 mm, Tosoh); solvent system, 0.1 m NH4OAc (0.05% trifluoroacetic acid)/acetonitrile (0.05% trifluoroacetic acid) (70:30); flow rate, 0.8 ml/min; detection, absorbance measurement at 312 nm with an SPD6A photodiode array detector. Product formation and substrate specificity were analyzed by LC-MS (LCMS-2010, Shimadzu) with the same conditions as in HPLC analysis except for the solvent system (acetonitrile/H2O/acetic acid (10∼25:89∼74:1)) and the flow rate (0.5 ml/min). When conversion rates were determined in the substrate specificity assay, the same solvent system and flow rate as in the LC-MS analysis were used in HPLC analysis. Stereoselectivity Assay of CYP80G2—The stereoselectivity of the CYP80G2 reaction was analyzed using normal-phase HPLC with the following conditions: column, CHIRALCEL OD-H (4.6 × 250 mm, Daicel Chemical Industries, Ltd., Japan); solvent system, hexane/2-propanol/diethylamine (80:20:0.1); flow rate, 0.8 ml/min; detection, absorbance measurement at 280 nm with an SPD6A photodiode array detector. HPLC samples were prepared by extracting the reaction mixture with ethyl acetate after an enzyme reaction for 30 min, and the ethyl acetate layer was directly subjected to normal-phase HPLC analysis. Determination of Kinetic Parameters—To determine the kinetic parameters of CYP80G2, the amount of corytuberine produced was estimated by the calibration curve of corytuberine (picomoles versus peak area) at 312 nm, which was drawn using the calibration curve of (R,S)-reticuline (picomoles versus peak area) at 283 nm, the absorption ratio of reticuline and corytuberine at 283 nm (1.00:0.93), and the absorption ratio of corytuberine between 283 and 312 nm (1.00:1.10). The data were fitted to the Michaelis-Menten equation by using a nonlinear least-square iterative method using KaleidaGraph (Synergy Software, Reading, PA). Three sets of kinetic parameters were obtained from three independent experiments and then simply averaged to yield the final estimates. The final estimates are shown with the standard errors for the three sets. Isolation of Cytochrome P450 cDNAs—Cultured C. japonica cells produce several kinds of isoquinoline alkaloids (Fig. 2). Because we speculated that magnoflorine and coptisine biosyntheses would include some of the oxidative reactions catalyzed by P450s, we searched for candidates of their biosynthetic P450 genes using 4032 redundant ESTs prepared from cultured C. japonica cells. A BLAST search (blastx) (www.ncbi.nlm.nih.gov/BLAST/) showed that these ESTs included P450 clones corresponding to CYP80B2, CYP719A1, and uncharacterized P450s (3, 5, and 17 clones, respectively). Because this EST library had multiple clones of berberine biosynthetic genes (CYP80B2 and CYP719A1), we expected that it would be useful for isolating a novel P450 gene involved in magnoflorine and coptisine biosyntheses. Further analysis of the uncharacterized P450s revealed that 17 clones were classified into 7 species, one of which, consisting of four clones, showed high homology to B. stolonifera CYP80A1 or E. californica CYP80B1 (the E values ranged from 1e-32 to 3e-50). Because both CYP80A1 and CYP80B1 were involved in isoquinoline alkaloid biosynthesis, we expected that this novel P450 species, which was temporarily designated CYP80A1-like, may also be responsible for isoquinoline alkaloid biosynthesis. Thus, its fulllength cDNA was isolated. Nucleotide Sequences and Predicted Amino Acid Sequences—Among the four EST clones of CYP80A1-like, the longest had 1517 nucleotides and still lacked the 5′-region of full-length P450 cDNA. To isolate the full-length of CYP80A1-like, 5′-RACE was conducted. Full-length CYP80A1-like cDNA, which was re-amplified from a cDNA library, contained 1700 nucleotides with an open reading frame of 486 amino acids (DDBJ/GenBank™/EMBL accession number AB288053) (Fig. 3). Although a blastx search showed that the amino acid sequence of CYP80A1-like was most similar to that of B. stolonifera CYP80A1 (53.1% identity), CYP80A1-like was classified into the CYP80G subfamily and designated CYP80G2 by the P450 nomenclature committee (c/o Dr. D. R. Nelson, University of Tennessee, Memphis, TN). The nomenclature committee informed us that CYP80G2 had quite high amino acid sequence similarity (85% identity) with CYP80G1, which has been isolated from ESTs of Aquilegia formosa × Aquilegia pubescens (Ranunculaceae) (GenBank™ accession numbers for its ESTs are DR912881.1, DR923264, and DT766891, and its amino acid sequence is available from Dr. D. R. Nelson). Although this high degree of homology indicated that CYP80G2 is an ortholog of CYP80G1, the function of CYP80G1 had not yet been clarified, and therefore we decided to characterize CYP80G2 (CYP80A1-like). Structural analysis showed that CYP80G2 had conserved eukaryotic P450 regions: a helix K region, an aromatic region, and a heme-binding region at the C-terminal end (Fig. 3). In addition, its N-terminal region contained hydrophobic domains corresponding to the membrane anchor sequences of microsomal P450 species, suggesting that CYP80G2 is localized in the endoplasmic reticulum. Notably, CYP80G2 had a unique amino acid substitution in a consensus amino acid sequence ((A/G)GX(D/E)T(T/S)) of the helix I region, which should be involved in interaction with the substrate and iron-bound oxygen (32Durst F. Nelson D.R. Drug Metabol. Drug Interact. 1995; 12: 189-206PubMed Google Scholar); i.e. CYP80G2 had a proline instead of alanine/glycine (underlined) at the position corresponding to Gly-248 in P450cam (33Poulos T.L. Finzel B.C. Gunsalus I.C. Wagner G.C. Kraut J. J. Biol. Chem. 1985; 260: 16122-16130Abstract Full Text PDF PubMed Google Scholar). The same substitution was also found in CYP80A1 (Fig. 3). Heterologous Expression of CYP80G2 in Yeast and Its Activity—To determine the function of CYP80G2, yeast expression plasmid for CYP80G2 was constructe