Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica Nobuhiro Ikezawa 1 , Kinuko Iwasa 2 and Fumihiko Sato 1 1 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan 2 Kobe Pharmaceutical University, Japan Higher plants produce structurally divergent chemi- cals, such as terpenoids, phenylpropanoids, and alka- loids [1]. Throughout human history, several plant materials have been used in natural medicines because of the pharmacological activities of these chemicals. Isoquinoline alkaloids are a large group of alkaloids, and include many pharmacologically useful compounds, 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 bio- synthesis 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 cyto- chrome 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 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)-cheilan- thifoline, (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. Abbreviations BBE, berberine bridge enzyme; CHS, (S)-cheilanthifoline synthase; MeJA, methyl jasmonate; 4¢-OMT, S-adenosyl- L-methionine : 3¢-hydroxy- N-methylcoclaurine 4¢-O-methyltransferase; P450, cytochrome P450; RNAi, RNA interference; (S)-THB, (S)-tetrahydroberberine; (S)-THC, (S)-tetrahydrocolumbamine; STS, (S)-stylopine synthase. FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS 1019 such as morphine. Despite their structural diversity, most isoquinoline alkaloids are found in limited and evolutionarily old taxonomic groups within the plant kingdom, i.e. Papaveraceae, Ranunculaceae, Berberida- ceae, Menispermaceae, and a few other families. Among the wide array of chemicals produced in these plant families, some are known to share, at least in part, common biosynthetic pathways, e.g. a key intermediate (S)-reticuline [1]. The Papaveraceae plant California poppy, Esch- scholzia californica, is a traditional medicinal plant of Native Americans, and has been well investigated because of the variety and pharmacological effects of its alkaloids. One of the alkaloids produced by this plant is the antimicrobial sanguinarine, which had been used as the component of toothpastes and mouthwashes. The biosynthetic pathways of sanguina- rine, as well as other benzophenanthridine-type alka- loids chelirubine and macarpine, have been completely elucidated at the enzyme level [2,3]. These highly oxid- ized chemicals are biosynthesized from two molecules of l -tyrosine via the central intermediate (S )-stylopine. (S)-Stylopine, which has two methylenedioxy groups in rings A and D of its protoberberine skeleton, is pro- duced by a three-step conversion from the key interme- diate (S)-reticuline; berberine bridge enzyme (BBE) catalyzes oxidative cyclization of the N-methyl moiety of (S)-reticuline to produce (S)-scoulerine [4,5], and two cytochrome P450s (S)-cheilanthifoline synthase (CHS; EC 1.14.21.2) and ( S )-stylopine synthase (STS; EC 1.14.21.1), sequentially form two methylenedioxy bridges from (S )-scoulerine via (S)-cheilanthifoline to (S)-stylopine [6,7] (Fig. 1). These two P450s (CHS and STS) have been studied in part by using microsomal fractions of cultured E. californica cells [7]. In that report, CHS and STS were found to be two independ- ent P450s, although they both catalyze methylenedioxy bridge-forming reactions using similar substrates [(S)-scoulerine and (S)-cheilanthifoline]. Because many P450s, especially those involved in the detoxification of xenobiotics, are known to have relatively broad sub- strate specificity [8], CHS and STS reactions might be catalyzed by a single P450 in different ways, or CHS and STS may be two homologous P450s. To clarify this point, we tried to isolate methylenedioxy bridge- forming P450 cDNA(s) from E. californica. Previously, we isolated the first methylenedioxy bridge-forming P450 (CYP719) cDNA, the protein of which catalyzed the conversion of (S)-tetrahydroco- lumbamine to (S)-tetrahydroberberine [(S)-canadine] in berberine biosynthesis, from cultured cells of Japanese goldthread Coptis japonica (Ranunculaceae) (Fig. 1) [9]. C. japonica produces a large amount of berberine, which has been used as a medicine, for example as a stomach tonic, and the biosynthesis of berberine has been intensively studied [10,11]. Because C. japonica cells also produce coptisine [12], oxidized stylopine, it has been proposed that stylopine is biosynthesized in C. japonica. However, C. japonica CYP719 did not show CHS activity, although it is possible that it had STS activity, which we were not able to check because of a lack of the substrate cheilanthifoline. Because no previous report has described stylopine biosynthesis in C. japonica, it is possible that a coptisine biosynthetic pathway in C. japonica may not be similar to the known stylopine biosynthetic pathway in E. californica. Because both CHS and STS catalyze methylenedioxy bridge-forming reactions using substrates quite similar to that of C. japonica CYP719 (Fig. 1), we speculated that the primary structures of CHS and STS in E. cali- fornica may be homologous to that of C. japonica CYP719. Based on this idea, we amplified cDNA frag- ments that were homologous to C. japonica CYP719 from single-stranded cDNAs prepared from cultured E. californica cells. Next, we isolated two full-length cDNAs and characterized the enzymological activity of their recombinant proteins produced in a S. cerevisiae expression system. Both of these CYP719 homologs (CYP719A2 and CYP719A3) showed methylenedioxy bridge-forming activity to convert cheilanthifoline to stylopine (STS activity), but no activity to convert scoulerine to cheilanthifoline (CHS activity). In addi- tion, they used scoulerine as the substrate to produce nandinine, which has a methylenedioxy bridge in ring A (2,3-position) of scoulerine. Based on their enzymological properties (substrate specificity and kin- etic parameters) and expression profiles, we discuss the physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis in E. californica. Results Isolation of cytochrome P450 cDNAs To examine the presence of C. japonica CYP719 homologs and their functions in stylopine biosynthesis in E. californica, cytochrome P450 (P450) cDNA frag- ments were amplified from single-stranded cDNAs, prepared from cultured E. californica cells, using the degenerate primers designed for the P450-conserved regions specific to C. japonica CYP719 [9]. After nested PCR, clear PCR products at  280 bp were obtained and subcloned into pT7Blue T-vector. Sequence analy- sis of two clones indicated that amplified cDNA frag- ments had an identical sequence that was highly homologous (73.2% identity for 280 bp PCR product) Stylopine synthase from Eschscholzia californica N. Ikezawa et al. 1020 FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS Fig. 1. Biosynthetic pathway for a variety of isoquinoline alkaloids. (S)-Scoulerine is an intermediate at the branch point leading to benzophe- nanthridine alkaloids in E. californica or berberine in C. japonica. The pathway from (S)-scoulerine to berberine is thought not to exist in E. californica, but is the main pathway in C. japonica (surrounded by dotted line). CYP80B1, (S)-N-methylcoclaurine 3¢-hydroxylase; 4¢-OMT, S-adenosyl- L-methionine:3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase; BBE, berberine bridge enzyme. N. Ikezawa et al. Stylopine synthase from Eschscholzia californica FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS 1021 to the corresponding region of C. japonica CYP719 cDNA (designated EcCYPA). To isolate a full-length clone of EcCYPA, 5¢- and 3¢-RACE were conducted. Although a full-length sequence of EcCYPA was obtained, another sequence obtained with 5¢-RACE showed a minor but distinct change from EcCYPA (named EcCYPB). A full-length clone of EcCYPB was also isolated with 5¢- and 3¢-RACE. Nucleotide sequences and predicted amino acid sequences Sequence analysis confirmed that the longest full- length EcCYPA carried 1916 nucleotides, with an ORF that encoded 495 amino acids (accession number AB126256), and full-length EcCYPB contained 1718 nucleotides, with an ORF that encoded 495 amino acids (accession number AB126257). EcCYPA and EcCYPB were also highly homologous to C. japonica CYP719 at the amino acid level (64.8 and 65.1% iden- tity, respectively), and the identity between EcCYPA and EcCYPB was 84.6%. These predicted amino acid sequences were classified into the same family as C. japonica CYP719 by the P450 nomenclature com- mittee (D.R. Nelson, University of Tennessee, Mem- phis, TN). According to the suggestion of the nomenclature committee, C. japonica CYP719 was renamed CYP719A1, and EcCYPA and EcCYPB were designated CYP719A3 and CYP719A2, respectively. Sequence analysis also showed that CYP719A2 and CYP719A3 had conserved eukaryotic P450 regions: a helix K region, an aromatic region, and a heme-bind- ing region at the C-terminal end (Fig. 2). In addition, their N-terminal regions contained hydrophobic domains that corresponded to the membrane anchor sequences of microsomal P450 species, which suggested that CYP719A2 and CYP719A3 were located in the endoplasmic reticulum. Notably, a conserved threonine (corresponding to Thr252 of P450cam), which plays a significant role in oxygen activation [13], has been replaced by serine in both CYP719A2 and CYP719A3 as in C. japonica CYP719A1 (Ser299 of both Fig. 2. Amino acid sequence alignment of the CYP719 family. Boxes indicate conserved regions of eukaryotic P450, which are the helix K region, aromatic region, and heme-binding region. The box with dotted line in the helix-K region indicates the conserved EXXR motif, which is canonical to all P450s. The arrows below the sequences indicate degenerate primers (Fw1, Fw2, and Rv1) used for the amplification of CYP719 homologous cDNA fragments from E. californica. The asterisk indicates the position of Ser299 of CYP719A2 and CYP719A3, which replaces the conserved threonine. CYP719A1, methylenedioxy bridge-forming enzyme from C. japonica (accession number AB026122). Stylopine synthase from Eschscholzia californica N. Ikezawa et al. 1022 FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS CYP719A2 and CYP719A3). This substitution is only found in a few species, such as Zea mays CYP88A1 (accession number U32579) and Nicotiana tabacum CYP92A2 (accession number X95342). Because the primary structures of CYP719A2 and CYP719A3 were so similar to that of C. japonica CYP719A1, these P450s were speculated to catalyze methylenedioxy bridge-forming reactions similar to C. japonica CYP719A1. Although C. japonica CYP719A1 is involved in berberine biosynthesis, it has been reported that E. californica does not produce a detectable level of berberine [14–17]. Thus, CYP719A2 and CYP719A3 appear to be involved in a biosynthetic pathway other than berberine biosynthesis. In contrast to C. japonica, E. californica produces a large amount of the benzophenanthridine alkaloid sanguinarine [14,15,18], the biosynthesis of which requires two consecutive methy- lenedioxy bridge-forming reactions from (S)-scoulerine via (S)-cheilanthifoline to (S)-stylopine, i.e. CHS and STS reactions. Because (S)-scoulerine and (S)-cheilanthi- foline are structurally similar to (S)-tetrahydrocolumb- amine [(S)-THC], the substrate of C. japonica CYP719A1 [9], we examined whether CYP719A2 and CYP719A3 could function as CHS or STS. Heterologous expression of CYP719A2 and CYP719A3 in S. cerevisiae and their enzymological activities CYP719A2 and CYP719A3 were heterologously expressed in S. cerevisiae to characterize their enzymo- logical activities. S. cerevisiae expression plasmids for CYP719A2 and CYP719A3 were constructed and introduced into the S. cerevisiae strain AH22. Because both CYP719A2 and CYP719A3 had putative endo- plasmic reticulum-localizing signals, microsomal frac- tions were prepared from recombinant S. cerevisiae cells, and their enzymatic activities were determined using LC-MS. Microsomal fractions prepared from transgenic S. cerevisiae cells of both CYP719A2 and CYP719A3 showed STS activity, i.e. they catalyzed methylenedi- oxy bridge-formation from (R,S)-cheilanthifoline to stylopine (Fig. 3). The formation of stylopine from cheilanthifoline was confirmed by direct comparison of the reaction product with standard stylopine. Although the concentration of ( R ,S )-cheilanthifoline used for enzyme assays was low (0.4 lm) due to its low availab- ility, recombinant CYP719A2 and CYP719A3 were able to convert it to stylopine, suggesting that both CYP719A2 and CYP719A3 function as STS. However, when (S)-scoulerine was used as the sub- strate, neither CYP719A2 nor CYP719A3 produced cheilanthifoline, but rather produced another product (see below). By contrast, C. japonica CYP719A1 does not utilize (S)-scoulerine [9], and did not utilize (R,S)- cheilanthifoline at a concentration of 0.4 l m (data not shown), despite its K m value of 0.27 lm toward (S)- THC [9]. These results suggested that members of the CYP719 family (CYP719A1–A3) have different sub- strate specificities and ⁄ or reaction activities. P450 natures of CYP719A2 and CYP719A3 The P450 natures of CYP719A2 and CYP719A3 were examined using reduced CO-difference spectra (Fig. 4). Whereas CYP719A2 expressed in S. cerevisiae cells showed a characteristic peak at 450 nm with a content of 30 pmol P450 per mg microsomal protein, the micro- somal fraction of CYP719A3 expressed in S. cerevisiae cells showed indistinguishable spectral pattern from that of control S. cerevisiae cells harboring empty plasmid. Because the expression level of CYP719A3 in S. cerevisiae cells was low, we characterized their P450 natures, particularly that of CYP719A3, based on their reaction dependencies on NADPH and oxygen using (S)-scoulerine as the substrate (Table 1). The absence of NADPH or removal of O 2 by the glucose ⁄ glucose oxidase ⁄ catalase system [7] clearly inhibited the CYP719A2 and CYP719A3 activities. Substrate specificity and affinity of CYP719A2 and CYP719A3 To investigate which of these P450s, CYP719A2 or CYP719A3, functions mainly as STS, their detailed substrate specificity was examined using several types of alkaloids harboring an ortho-methoxyphenol moiety at 10 lm (Fig. 5). LC-MS analysis showed that CYP719A2 and CYP719A3 could convert (S)-scouler- ine to a new product with a reduction of 2 m ⁄ z. This new product was suggested to be nandinine based on its total m ⁄ z and fragment ion patterns (data not shown). Nandinine is a scoulerine derivative with the methylenedioxy bridge in ring A (2,3-position), whereas cheilanthifoline is a derivative with the methylenedioxy bridge in ring D (9,10-position). Because neither CYP719A2 nor CYP719A3 catalyzed the methylenedioxy bridge-formation in ring D of scoulerine, they should strictly recognize ring A of the substrate. Whereas CYP719A3 also converted (S)-THC to (S)-tetrahydroberberine [(S)-THB], CYP719A2 did not (data not shown). Neither CYP719A2 nor CYP719A3 reacted with any of seven other compounds [columbamine, (R,S)-reticuline, (R,S)- norreticuline, (S)-N-methylcoclaurine, (S)-coclaurine N. Ikezawa et al. Stylopine synthase from Eschscholzia californica FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS 1023 (R,S)-6-O-methylnorlaudanosoline, and magnoflorine] to make their corresponding products with a methylen- edioxy bridge. Also, neither CYP719A2 nor CYP719A3 catalyzed methylenedioxy bridge-formation with a (S)-scoulerine derivative, which was produced from (S)-scoulerine by S-adenosyl-l-methionine : coclaurine TIC m/z=326 m/z=324 min 10 20 30 (R,S)-cheilanthifoline; m/z=326 H 3 CO CO HO HO N O O 0 10 8 6 4 2 0 height ( 10 6 ) A TIC m/z=326 m/z=324 min 10 20 30 reaction product 0 10 8 6 4 2 0 height ( 10 6 ) B TIC m/z=326 m/z=324 min reaction product 10 20 30 0 10 8 6 4 2 0 height ( 10 6 ) C TIC m/z=326 m/z=324 min 10 20 30 (R,S)-stylopine; m/z=324 O O N O O 0 10 8 6 4 2 0 height ( 10 5 ) D Fig. 3. LC-MS analysis of reaction products of CYP719A2 and CYP719A3 using (R,S)-ch- eilanthifoline as the substrate. Vector control reaction (A), CYP719A2 reaction (B), CYP719A3 reaction (C), and authentic (R,S)-stylopine (D) are shown. TIC, total ion chromatogram. Stylopine synthase from Eschscholzia californica N. Ikezawa et al. 1024 FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS N-methyltransferase [19] and was predicted to be N-methylated (S)-scoulerine. Because the above results suggested that (R,S)-chei- lanthifoline and (S)-scoulerine would be competitive substrates for CYP719A2 and CYP719A3, we exam- ined the reaction specificity using a mixture of these compounds at 0.4 lm each. As shown in Fig. 6, CYP719A2 exclusively used (R,S)-cheilanthifoline as a substrate to produce stylopine. By contrast, CYP719A3 converted (R,S)-cheilanthifoline and (S)-scoulerine to stylopine and nandinine at a compar- able level (Fig. 6). Furthermore, when (R,S)-cheilan- thifoline and (S)-THC were used as mixed substrates at 0.4 lm each, CYP719A3 showed comparable methy- lenedioxy bridge-forming activities with both substrates (Fig. 6). These results indicated that CYP719A3 had broader substrate specificity than CYP719A2, although only the ring A (2,3-position) of the substrates was converted. Next, the substrate affinities of CYP719A2 and CYP719A3 were determined to examine their functions in greater detail. Enzyme activity was quantified by HPLC using (R,S)-scoulerine (for CYP719A2) or (S)-scoulerine (for CYP719A3) as the substrates, due to the low availability of (R,S)-cheilanthifoline. Both P450s showed Michaelis–Menten-type reaction kinetics when the substrate concentration was varied. The kin- etic parameters, K m and V max , of CYP719A2 were esti- mated to be 32 ± 7.1 lm and 0.43 ± 0.04 pmol productÆmin )1 Æpmol )1 P450, respectively, whereas those of CYP719A3 were estimated to be 0.54 ± 0.03 lm and 4.5 ± 0.03 pmol productÆmin )1 Æmg )1 microsomal protein. These differences in the K m values for scouler- ine explain the different reactivities of CYP719A2 and CYP719A3 for the mixture of cheilanthifoline and scoulerine (Fig. 6), because both CYP719A2 and CYP719A3 catalyzed (R,S)-cheilanthifoline compar- ably at a substrate concentration of 0.4 lm (Fig. 3). Expression of CYP719A2 and CYP719A3 genes in E. californica plants: tissue specificity and their response to methyl jasmonate (MeJA) We examined the accumulation of mRNA of CYP719A2 and CYP719A3 in E. cali fornica plant tissues (leaf, stem, and root) in comparison with three stylopine biosynthetic genes, CYP80B1 [(S)-N-methylcoclaurine 3¢-hydroxylase] [20], BBE [4,5], and S-adenosyl-l-methionine : 3¢-hydroxy- N-meth ylcoclaurine 4¢-O-methyltransferase (4¢-OMT) [21, 21a] (Fig. 7). As a r esult, all of the genes e xamined showed similar expression patterns; the expression in root was greater than that in the leaf and stem, which suggests that both CYP719A2 and CYP719A3 are involved in alkaloid Absorbance (10 -3 ) 4 -4 0 400 500450 Wavelen g th (nm) Absorbance (10 -3 ) 4 -4 0 400 500450 Wavelen g th (nm) AB Fig. 4. Reduced CO-difference spectra of CYP719A2 and CYP719A3 heterologously expressed in S. cerevisiae. Reduced CO-difference spectra were recorded using microsomal fractions of recombinant S. cerevisiae (1 mg proteinÆmL )1 ). Solid line is the spectrum of the microsomal fraction from CYP719A2-expressing S. cerevisiae (A) or CYP719A3-expressing S. cerevisiae (B). Dotted line is the spectrum of the micro- somal fraction from control S. cerevisiae transformed with empty plasmid. Table 1. Methylenedioxy bridge-forming activity of CYP719A2 and CYP719A3 toward (S)-scoulerine without NADPH or oxygen. The reaction was carried out under standard assay conditions except for the amounts of enzyme preparations (200 n M P450 for CYP719A2 or 1.1 lgÆlL )1 of microsomal protein for CYP719A3) after removal of NADPH or oxygen with glucose ⁄ glucose oxidase. U, units. Addition Relative activity CYP719A2 % CYP719A3 % None 100 100 – NADPH 0 0 40 m M glucose +5 U glucose oxidase +10 U catalase 04 40 m M glucose + boiled glucose oxidase +10 U catalase 100 105 N. Ikezawa et al. Stylopine synthase from Eschscholzia californica FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS 1025 biosynthesis (Fig. 7). Notably, CYP719A3 showed extre- mely high expression in root compared with the other tis- sues ( 30 times h igher than in leaf), whereas other genes only showed a moderate increase in expression in root ( 3–6 times higher t han i n l eaf). W hen t he probable relat- ive expression levels of CYP719A2 and CYP719A3 were Fig. 5. Chemical structures of compounds tested as potential substrates for CYP719A2 and CYP719A3. The apparent natural sub- strate (R,S)-cheilanthifoline is boxed. Iso- quinoline alkaloids with an ortho- methoxyphenol moiety were tested as potential substrates. The structure of ‘N-methylated (S)-scoulerine’ is speculated based on the reaction mechanism of S-adenosyl- L-methionine:coclaurine N-methyltransferase. Stylopine synthase from Eschscholzia californica N. Ikezawa et al. 1026 FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS estimated from their copy numbers in the same samples using standard curves drawn w ith plasmids ( pT7Blue- based constructs containing each gene) with quantitative RT-PCR, the calculation indicated that the probable relat- ive expression level of CYP719A3 against CYP719A2 was  4 times higher in root, w hereas it was < 2 t imes lowe r in leaf and stem (data not shown). Because the expression of CYP80B1, BBE, 4 ¢-OMT, CYP719A2, and CYP719A3 was all the highest in root, and sanguinarine is found exclusively in r oot (data n ot s hown), root is likely the main organ for the biosynthesis and storage of sanguinarine in E. californica. These results suggested that CYP719A3 might contribute more to stylopine biosynthesis than CYP719A2 in root, whereas the physiological functions of CYP719A2 and CYP719A3 need to be characterized fur- ther. The effects of MeJA on the expression of CYP719A2 and CYP719A3 in E. californica seedlings were examined, because MeJA has been reported to induce the biosynthesis of various plant secondary metabolites [22–26], including benzophenanthridine alkaloid bio- synthesis in E. californica [22]. MeJA also induced the expression of the CYP80B1 and BBE genes [10], and the activities of CHS and STS [27]. Our results indica- ted that treatment with MeJA increased the expression of all five genes (CYP719A2, CYP719A3, CYP80B1, BBE, and 4¢-OMT), which suggests the cooperative regulation of these biosynthetic genes (Fig. 8). How- ever, detailed characterization of their induction kinet- ics indicated minor but still marked differences; CYP719A2, CYP719A3, CYP80B1, and 4¢-OMT tran- scripts accumulated rapidly to reach the maximum level within 12 h, whereas the BBE transcript showed a slower and prolonged increase over 48 h (Fig. 8). Concerning the differences between CYP719A2 and CYP719A3 transcripts, although their increases showed A B C D E F Fig. 6. CYP719A2 and CYP719A3 have different substrate selectivities toward (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumb- amine. The reaction mixtures contained two substrates at 0.4 l M each. For the (R,S)-cheilanthifoline and (S)-scoulerine assay, vector control reaction (A), CYP719A2 reaction (B), CYP719A3 reaction (C), and authentic (R,S)-stylopine (D) are shown. For the (R,S)-cheilanthifoline and (S)-tetrahydrocolumbamine assay, CYP719A3 reaction (E) and authentic (R,S)-tetrahydroberberine (F) are shown. Reaction products (1) and (3) showed the same retention times and m ⁄ z-values as authentic (R,S)-stylopine and (R,S)-tetrahydroberberine, respectively, whereas the reaction product (2) was suggested to be nandinine by its total m ⁄ z and fragment ion patterns (data not shown). N. Ikezawa et al. Stylopine synthase from Eschscholzia californica FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS 1027 similar kinetics, only CYP719A2 transcript showed a rather constant expression level even at 48 h (Fig. 8). This result may reflect the physiologically different functions of CYP719A2 and CYP719A3. Discussion We isolated two full-length P450 cDNAs (CYP719A2 and CYP719A3) using degenerate primers, designed for C. japonica CYP719A1, from cultured E. californica cells (Fig. 2). The primary structures of CYP719A2 and CYP719A3 showed high similarity to C. japonica CYP719A1 (65.1 and 64.8% identity, respectively). Their recombinant proteins produced in S. cerevisiae showed the activity of STS, which catalyzes the methy- lenedioxy bridge-forming reaction from cheilanthifoline to stylopine (Fig. 3). By contrast, neither of them, nor C. japonica CYP719A1, showed the activity of CHS, which catalyzes the methylenedioxy bridge-forming reaction from scoulerine to cheilanthifoline (Fig. 1). These results indicate that another enzyme is needed for CHS activity in stylopine biosynthesis, and supports a previous report that CHS and STS are independent P450s [7]. Analysis of substrate specificity showed that CYP719A2 and CYP719A3 catalyzed methylenedioxy bridge-formation only in ring A of their substrates, e.g. the conversion of scoulerine to nandinine (Fig. 5). Our results indicate that members of the CYP719 family have strict substrate specificity for ring A (2,3-position) A D B E C Fig. 7. Expression profiles of CYP719A2 and CYP719A3 genes in plant tissues of E. cali- fornica. Total RNA (1 lg) prepared from E. californica plant tissues (leaf, stem, and root) was used for DNase I treatment and reverse transcription. Transcript levels of CYP80B1 (A), BBE (B), 4¢OMT (C), CYP719A2 (D), and CYP719A3 (E) were determined using quantitative RT-PCR with quadruple measurements. The relative expression level shows the values standard- ized by that of leaf as 1. The error bar indi- cates the standard error of the mean. Stylopine synthase from Eschscholzia californica N. Ikezawa et al. 1028 FEBS Journal 274 (2007) 1019–1035 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... puzzle in alkaloid biosynthesis in E californica, because CYP719A2 and CYP719A3 produced (S)-nandinine from (S)-scoulerine in vitro; indeed, CYP719A3 converted (S)-scoulerine to (S)-nandinine with high affinity (Km ¼ 0.54 lm) (Fig 6) Previous reports have shown that (S) -stylopine is synthesized via (S)-cheilanthifoline, not (S)-nandinine [7,30,31] If we assume that (S)-nandinine is produced in E californica, ... by using a calibration curve of (S)-scoulerine (pmol versus peak area) at 285 nm To determine the kinetic parameters of CYP719A3, the amount of nandinine produced was estimated by using the calibration curve of (S)-scoulerine (pmol versus peak area) and the absorption ratio of (S)-scoulerine and (S)-nandinine at 285 nm (1.00 : 0.93) The data were fitted to the Michaelis–Menten equation by a nonlinear... (R,S)-cheilanthifoline and (S)-scoulerine, and CYP719A3 used (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-THC In addition, CYP719A2 and CYP719A3 showed considerably different substrate affinities toward scoulerine from each other; the Km values of CYP719A2 and CYP719A3 were 32.3 lm for (R,S)-scoulerine and 0.54 lm for (S)-scoulerine, respectively Because (R,S)-scoulerine was expected to contain equal amounts of. .. (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-THC (Fig 6) These results suggested that CYP719A2 might function only as STS, whereas CYP719A3 might be involved in the biosynthesis of stylopine and in other biosyntheses However, analysis of their expression in E californica plant tissues showed that CYP719A3 mRNA was specifically accumulated in root ( 30 times higher than in leaf) (Fig 7), and the probable... Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications Annu Rev Plant Physiol Plant Mol Biol 52, 29–66 Sato F & Yamada Y (2007) Engineering medicinal compounds in cell cultures In Bioengineering and Molecular Biology of Plant Pathways, Vol 1 (Bohnert HJ & Nguyen HT, eds), Elsevier, Amsterdam In press Sato F & Yamada Y (1984) High berberine-producing... (1975) Biosynthesis Part XXII The origin of chelidonine and of other alkaloids derived from the tetrahydroprotoberberine skeleton J Chem Soc Perkin I, 1147–1156 Takao N, Iwasa K, Kamigauchi M & Sugiura M (1976) Studies on the alkaloids of Papaveraceous plants XXV Biosynthesis of the alkaloids of Corydalis incisa Pers and Chelidonium majus L incorporations of tetrahydroprotoberberines, N-methosalts of tetrahydroprotoberberines,... 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica J Biol Chem 275, 23398–23405 Inui T, Tamura K, Fujii N, Morishige T & Sato F (2007) Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia california Plant Cell Physiol, doi:... need for a catalyst from (S)-nandinine to (S) -stylopine However, nandinine could not be detected when the extracts of E californica plant tissues were analyzed using LC-MS (data not shown), which suggests that (S)-nandinine, if produced, would be metabolized immediately to other compound(s) However, it is possible that CYP719A3 could not use (S)-scoulerine in vivo; (S)-scoulerine might be consumed by CHS... separation of the common alkaloids of the berberine group; the isolation and characterization of columbamine Lloydia (Cinci) 28, 73–83 40 Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive Stylopine synthase from Eschscholzia californica 41 42 43 44 45 46 47 multiple sequence alignment through sequence weighting, position-specific gap penalties and weight... ring D (9,10-position) of scoulerine This suggests that strategies different from the one used in this study will be needed to clone CHS cDNA, e.g the overall screening of P450 genes using a cDNA library, prepared from MeJA-treated E californica cells, would be effective, because CHS activity has been reported to increase in response to MeJA together with those of other P450s involved in sanguinarine . Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica Nobuhiro. 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