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Fumihiko Sato - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of cheilanthifoline and Stylopine synthase genes from chelidonium majus
Plant and Cell Physiology, 2017Co-Authors: Mahdi Yahyazadeh, Fumihiko Sato, Purwanto Ratmoyo, Florian Bittner, Dirk SelmarAbstract:: The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a Stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to Stylopine by the action of a Stylopine synthase (c1128). Based on the well-known instability of Stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and Stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by Stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and Stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.
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Efficient microbial production of Stylopine using a Pichia pastoris expression system
Scientific Reports, 2016Co-Authors: Kentaro Hori, Shunsuke Okano, Fumihiko SatoAbstract:Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of ( S )-reticuline, we attempted to produce Stylopine from ( S )-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5) and Stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast ( Pichia pastoris ) and both a “consolidated” system with all genes expressed in one cell and a “co-culture” system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to Stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of Stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of ( R, S )-reticuline (375 nmoles of ( S )-reticuline). The advantages and drawbacks of the “consolidated” system and the “co-culture” system are discussed.
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Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.
Phytochemistry, 2012Co-Authors: Tomoya Takemura, Nobuhiro Ikezawa, Kinuko Iwasa, Fumihiko SatoAbstract: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.
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molecular cloning and characterization of methylenedioxy bridge forming enzymes involved in Stylopine biosynthesis in eschscholzia californica
FEBS Journal, 2007Co-Authors: Nobuhiro Ikezawa, Kinuko Iwasa, Fumihiko SatoAbstract:(S)-Stylopine is an important intermediate in the biosynthesis of benzophenanthridine 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 Chem278, 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 Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae 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 expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in Stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three Stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in Stylopine biosynthesis are discussed.
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Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells.
Journal of Biological Chemistry, 2003Co-Authors: Nobuhiro Ikezawa, Masaru Tanaka, Masanori Nagayoshi, Raku Shinkyo, Toshiyuki Sakaki, Kuniyo Inouye, Fumihiko SatoAbstract:Abstract Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3′-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-Stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.
Eva Táborská - One of the best experts on this subject based on the ideXlab platform.
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Seasonal variation in alkaloid composition and antiproliferative activity of Stylophorum lasiocarpum (Oliv.) Fedde
Chemical Papers, 2015Co-Authors: Kristýna Šebrlová, Iva Slaninová, Ondřej Vymazal, Jana Kantorová, Eva TáborskáAbstract:Stylophorum lasiocarpum (Oliv.) Fedde (Papaveraceae) belongs to traditional Chinese medicine herbs but there was minimal information on the content of alkaloids in this plant. Extracts from the aerial part and roots were examined by liquid chromatography with UV and mass spectrometric detection, with nineteen alkaloids identified. Changes in alkaloid content over the entire vegetation period of a one- and two-year old plant were studied. The protoberberine alkaloids, coptisine and Stylopine, were found to be the main substances in extracts of the aerial part irrespective of the plant’s age and time of harvest. Variable amounts of protopine, sanguinarine, chelerythrine, chelirubine, macarpine, chelilutine and berberine were also recorded in the aerial part. The roots contained significantly larger quantities of all alkaloids than the aerial part with the levels of most alkaloids varying from May to October, peaking in the middle of the vegetation period. Coptisine was the dominant alkaloid in all samples. The antiproliferative activities of the root extract and of seven individual alkaloids were tested on A375 human malignant melanoma cells. The significant dose-dependent toxicity of the root extract was attributed largely to the quaternary benzo[ c ]phenanthridine alkaloids, macarpine and sanguinarine.
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Isoquinoline alkaloids: a 15N NMR and x-ray study. Part 2
Magnetic Resonance in Chemistry, 2002Co-Authors: Radek Marek, Jiří Slavík, Eva Táborská, Jaromír Marek, Jiří Dostál, Roger DommisseAbstract:Continuing our systematic 15N NMR study of isoquinoline alkaloids, we report a contribution extending our previous paper. The 15N NMR chemical shifts and 15N,1H long-range coupling pathways of tertiary and quaternary isoquinoline alkaloids of several constitutional types are presented. The selected compounds belong to the protoberberine, proaporphine, pavinane, rhoeadine and phtalideisoquinoline classes of alkaloids and were investigated by gradient-selected inverse-detected multiple bond correlation experiments (GHMBC and GSQMBC). In addition, x-ray data and the principal geometric parameters of Stylopine, mecambridine, norchelerythrine, isothebaine and mecambrine are reported and discussed.
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Alkaloids and organic acids content of eight Fumaria species
Phytochemical Analysis, 1999Co-Authors: J. Sousek, Eva Táborská, D. Guédon, T. Adam, Hana Bochořáková, I. Valka, Vilím ŠimánekAbstract:The isoquinoline alkaloids adlumiceine, adlumidiceine, coptisine, corytuberine, cryptopine, fumaricine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, palmatine, parfumine, protopine, sinactine, Stylopine, and N-methylStylopine were determined by reversed phase high performance liquid chromatography in Fumaria agraria, F. capreolata, F. densiflora, F. muralis, F. officinalis, F. parviflora, and F. vaillantii. Organic acids, namely citric, coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic, protocatechuic acid and caffeic acid (and its methylester) were identified by gas chromatography—mass spectrometry. The total content of phenolic constituents was quantified by a colorimetric method using a phosphomolybdic–phosphotungstic reagent. The chemotaxonomic significance of the results is discussed. Copyright © 1999 John Wiley & Sons, Ltd.
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FUMARIA DENSIFLORA DC. ALKALOIDS
Collection of Czechoslovak Chemical Communications, 1996Co-Authors: Eva Táborská, J. Sousek, Hana Bochořáková, Petr Sedmera, Ctirada Vavrečková, Vilím ŠimánekAbstract:Twenty isoquinoline alkaloids were identified in the aerial part of Fumaria densiflora DC. In addition to bicuculline, densiflorine, fumaritine, fumariline, scoulerine, coptisine, cryptopine, parfumine, protopine, and sinactine which have been found previously, the following alkaloids were isolated for the first time from this species: adlumine, fumaricine, fumaritridine, fumarophycine, fumarofine, corytuberine, cis - N -methylstylopinium iodide, Stylopine, and two new 1-benzylisoquinoline alkaloids, viz. fumaflorine and fumaflorine methyl ester. The cytoprotectivity of extracts of F . densiflora and F. officinalis , and that of protopine, cryptopine, and parfumine was tested on primary cultures of rat hepatocytes intoxicated with carbon tetrachloride. The cytoprotective effect of the extracts is associated with the alkaloids.
Nobuhiro Ikezawa - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.
Phytochemistry, 2012Co-Authors: Tomoya Takemura, Nobuhiro Ikezawa, Kinuko Iwasa, Fumihiko SatoAbstract: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.
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molecular cloning and characterization of methylenedioxy bridge forming enzymes involved in Stylopine biosynthesis in eschscholzia californica
FEBS Journal, 2007Co-Authors: Nobuhiro Ikezawa, Kinuko Iwasa, Fumihiko SatoAbstract:(S)-Stylopine is an important intermediate in the biosynthesis of benzophenanthridine 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 Chem278, 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 Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae 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 expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in Stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three Stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in Stylopine biosynthesis are discussed.
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Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells.
Journal of Biological Chemistry, 2003Co-Authors: Nobuhiro Ikezawa, Masaru Tanaka, Masanori Nagayoshi, Raku Shinkyo, Toshiyuki Sakaki, Kuniyo Inouye, Fumihiko SatoAbstract:Abstract Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3′-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-Stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.
Kyu-yun Chai - One of the best experts on this subject based on the ideXlab platform.
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Stylopine fromChelidonium majus inhibits LPS-Induced inflammatory mediators in RAW 264.7 cells
Archives of Pharmacal Research, 2004Co-Authors: Seon Jang, Sang Jin An, Han Gil Choi, Byung Hun Jeon, Hun-taeg Chung, Kyu-yun ChaiAbstract:Stylopine is a major component of the leaf of Chelidonium majus L. (Papaveraceae), which has been used for the removal of warts, papillomas and condylomas, as well as the treatment of liver disease, in oriental countries. Stylopine per se had no cytotoxic effect in unstimulated RAW 264.7 cells, but concentration-dependently reduced nitric oxide (NO), prostaglandin E_2 (PGE_2), tumor necrosis factor-a (TNF-α) and interleukin-1 β (IL-1β), and the IL-6 production and cyclooxygenase-2 (COX-2) activity caused by the LPS stimulation. The levels of inducible nitric oxide synthase (iNOS) and COX-2 protein expressions were markedly suppressed by Stylopine in a concentration dependent manner. These results suggest that Stylopine suppress the NO and PGE_2 production in macrophages by inhibiting the iNOS and COX-2 expressions. These biological activities of Stylopine may contribute to the anti-inflammatory activity of Chelidonium majus.
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Stylopine from chelidonium majus inhibits lps induced inflammatory mediators in raw 264 7 cells
Archives of Pharmacal Research, 2004Co-Authors: Seon Il Jang, Sang Jin An, Han Gil Choi, Byung Hun Jeon, Hun-taeg Chung, Kyu-yun ChaiAbstract:Stylopine is a major component of the leaf ofChelidonium majus L. (Papaveraceae), which has been used for the removal of warts, papillomas and condylomas, as well as the treatment of liver disease, in oriental countries. Stylopineper se had no cytotoxic effect in unstimulated RAW 264.7 cells, but concentration-dependently reduced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-α) and interleukin-1 β (IL-1β), and the IL-6 production and cyclooxygenase-2 (COX-2) activity caused by the LPS stimulation. The levels of inducible nitric oxide synthase (iNOS) and COX-2 protein expressions were markedly suppressed by Stylopine in a concentration dependent manner. These results suggest that Stylopine suppress the NO and PGE2 production in macrophages by inhibiting the iNOS and COX-2 expressions. These biological activities of Stylopine may contribute to the anti-inflammatory activity ofChelidonium majus.
Dirk Selmar - One of the best experts on this subject based on the ideXlab platform.
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Impact of drought and salt stress on the biosynthesis of alkaloids in Chelidonium majus L.
Phytochemistry, 2018Co-Authors: Yahyazadeh, Rieke Meinen, Robert Hänsch, Sara Abouzeid, Dirk SelmarAbstract:Abstract When plants are exposed to various stress situations, their alkaloid concentration frequently is enhanced. This well-known phenomenon is presumably due to a passively enhanced rate of biosynthesis, caused by greatly elevated concentrations of NADPH in stressed plants. Here, we used Chelidonium majus L. plants, which accumulate high concentrations of dihydrocoptisine in their leaves, to study the impact of drought and salt stress on the biosynthesis and accumulation of alkaloids. In comparison to well-watered controls, in the transcriptome of the gene encoding the key enzyme in alkaloid biosynthesis, Stylopine synthase, is enhanced in stressed C. majus plants. If we presuppose that increased transcript levels correlate with increased enzymatic activity of the gene products, these data indicate, for the first time, that stress-related increases in alkaloid concentration might not only be caused by the well-known stress-related passive shift, but may also be due to an enhancement of enzymatic capacity.
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cloning and characterization of cheilanthifoline and Stylopine synthase genes from chelidonium majus
Plant and Cell Physiology, 2017Co-Authors: Mahdi Yahyazadeh, Fumihiko Sato, Purwanto Ratmoyo, Florian Bittner, Dirk SelmarAbstract:: The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a Stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to Stylopine by the action of a Stylopine synthase (c1128). Based on the well-known instability of Stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and Stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by Stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and Stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.
