The Plant Journal (2009) 60, 56–67 doi: 10.1111/j.1365-313X.2009.03937.x Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L)
Silke Pienkny, Wolfgang Brandt, Ju¨ rgen Schmidt, Robert Kramell and Jo¨ rg Ziegler†,* Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany
Received 13 April 2009; accepted 8 May 2009; published online 13 July 2009. *For correspondence (fax +1 403 289 9311; e-mail [email protected]). †Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada.
SUMMARY
The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species-specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full-length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O-methyltransferase protein family. It was related to O-methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6-O-methyltransferase. However, rather than
methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a Km of 44 lM using S-adenosyl-L-methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7–O-methyltransferase (N7OMT). This enzyme represents a novel O- methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expres- sion of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.
Keywords: benzylisoquinoline alkaloids, O-methyltransferase, Papaver, opium poppy, papaverine biosynthe- sis, secondary metabolism.
INTRODUCTION Benzylisoquinoline alkaloids (BIAs) constitute of a group of its inhibitory effect on phosphodiesterases (Boswell-Smith natural products with diverse structures, which are all et al., 2006). derived from the amino acid tyrosine. So far, about 2500 The biosynthesis of all BIAs begins with the conden- compounds have been identified, several of which exhibit sation of the tyrosine-derived compounds dopamine and important pharmaceutical properties. Morphine is one of the p-hydroxyphenylacetaldehyde by norcoclaurine synthase, most powerful analgesics (Goodman et al., 2007), while its yielding the basic tetrahydrobenzylisoquinoline (S)-norco- precursor, codeine, is widely used as an antitussive (Chung, claurine. Initial modifications include 6- and 4¢-O-methyla- 2005). The benzophenanthridine sanguinarine and the pro- tions, N-methylation and 3¢-hydroxylation that lead to toberberine alkaloid berberine exert potent antimicrobial (S)-reticuline (Figure 1). This central intermediate is exten- activities (Colombo and Bosisio, 1996). The simple benzyl- sively modified in subsequent pathways leading to the isoquinoline papaverine is used as a vasodilator for treat- majority of benzylisoquinoline structures. Oxidative C–C ment of vasospasms (Brisman et al., 2006) and erectile bond formation between the N-methyl group and the ortho- dysfunction (Thomas, 2002), and as a smooth muscle carbon of the benzyl moiety results in berberine bridge relaxant (Sato et al., 2007). All these effects are attributed to formation and initiates the biosynthesis of protoberberine
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Figure 1. Benzylisoquinoline alkaloid biosynthesis. Conversions which are catalyzed by more than one enzyme are indicated as double arrows. Question marks denote steps not unequivocally resolved yet. 4¢OMT, (S)- 3¢hydroxy N-methylcoclaurine 4¢O-methyltransferase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; CNMT, (S)-coclaurine N-methyltransferase; CoOMT, columbamine O-methyltransferase; COR1, codeinone reductase 1; CYP80B3, (S)-N-methyl coclaurine 3¢hydroxylase; HPAA, p-hydroxy phenylacetaldehyde; NCS, (S)-norcoclaurine synthase; SalAT, 7(S)-salutaridinol 7-O-acetyltransferase; SalR, salutaridine reductase; SalSyn, salutaridine synthase; SOMT, (S)-scoulerine 9-O-methyltransferase; STOX, (S)-tetrahydroprotoberberine oxidase; TYDC, tyrosine decarboxylase. Protoberberine alkaloids are boxed in dark grey, promorphinan and morphinan alkaloids in light grey.
and benzophenanthridine alkaloids. The inversion of stereo- additional step. Up to now, neither cDNAs nor enzyme chemistry from (S)-reticuline to its (R)-enantiomer and activities which might support one or the other metabolic subsequent carbon–carbon phenol coupling leads to pro- route have been reported for papaverine biosynthesis. morphinan and morphinan alkaloids. Both pathways have O-Methylation is a common theme in the biosynthesis of been extensively investigated in the past years and several BIAs and several cDNAs encoding proteins that catalyze cDNAs coding for biosynthetic enzymes have been obtained these reactions have been obtained (Figure 1). Norcoclau- (Ziegler and Facchini, 2008). This is in contrast to the rine 6-O-methyltransferase (6OMT) and 3¢-hydroxy-N-meth- biosynthesis of papaverine, for which the pathway is not ylcoclaurine 4¢-O-methyltransferase (4¢OMT) both act in the completely understood. Tracer experiments suggested that early pathway up to reticuline (Frick and Kutchan, 1999; it might be derived from norreticuline or nororientaline Morishige et al., 2000; Ounaroon et al., 2003; Ziegler et al., (Brochmann-Hanssen et al., 1971, 1975). Accordingly, two 2005; Inui et al., 2007). Scoulerine 9-O-methyltransferase additional O-methylations must occur, as well as the dehy- (SOMT) as well as columbamine O-methyltransferase (CoO- drogenation of the heterocyclic ring. The results from the MT) catalyze reactions in the protoberberine branch of feeding experiments revealed that dehydrogenation occurs the pathway (Takeshita et al., 1995; Morishige et al., 2002). after methylation of all hydroxyl groups. However, the Reticuline 7-O-methyltransferase (7OMT) leads to laudanine, participation of reticuline as an intermediate compound in which might serve as an intermediate in papaverine biosyn- papaverine biosynthesis could not be completely ruled out. thesis assuming N-demethylation at a later stage (Ounaroon This would require the demethylation of the nitrogen as an et al., 2003). All of these OMTs belong to the class II type
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O-methyltransferases which use S-adenosyl-L-methionine of 4¢OMT (Ziegler et al., 2005, 2006). In this paper, we report (SAM) as the methyl donor, and do not require a metal ion the characterization of a third cDNA present in that cluster as for activity. They exhibit considerable amino acid identity a novel, highly specific class II type OMT, norreticuline 7-O- of at least 30% with 6OMT and 4¢OMT showing a close methyltransferase. The possible involvement of this enzyme relationship of more than 50% identity. They exhibit high in papaverine biosynthesis is discussed. substrate specificity with the highest catalytic efficiency toward their natural substrate. Nevertheless, selected mod- RESULTS ifications in methylation patterns of the alkaloid substrate Detection and sequence analysis of EST A21G11 may be tolerated by most OMTs (Takeshita et al., 1995; Frick and Kutchan, 1999; Morishige et al., 2000, 2002; Ounaroon Large-scale transcriptome and alkaloid analysis in Papaver et al., 2003; Ziegler et al., 2005; Inui et al., 2007). Remark- species yielded 69 cDNAs showing higher expression in six ably, none of the OMTs accepts the in vivo substrate of P. somniferum varieties compared with 15 other Papaver another OMT, suggesting a strict order in which O-methyl- species (Ziegler et al., 2006). The comparison between ation occurs in benzylisoquinoline biosynthesis. Addition- P. somniferum and the other Papaver species was chosen ally, they show strong regiospecificity with their natural because P. somniferum was bred for increased alkaloid substrate. However, depending on the provided substrate, accumulation. Additionally, it is the only species among the 7OMT from Papaver somniferum has been shown to selected ones which was reported to produce morphinan perform O-methylations at the 6 and 4¢ position as well as alkaloids as well as papaverine and noscapine (Shulgin and double methylations, and 4¢OMT from Eschscholzia califor- Perry, 2002; Ziegler et al., 2005, 2006). The differentially nica and Coptis japonica exhibited traces of 6-O-methylating expressed cDNAs were distributed in six gene expression activities (Ounaroon et al., 2003; Inui et al., 2007). clusters. Sequence analysis showed that no function could In recent publications we have reported comparative be assigned to 62% of the cDNAs, based on database com- transcript and alkaloid profiling in Papaver species showing parison. Eight coded for proteins presumably involved that most enzymes implicated in BIA metabolism show in secondary metabolism, including six P450-dependent coordinate expression and are present in one gene expres- monooxygenases. Additionally, all cDNAs already known to sion cluster exhibiting higher expression in plants which encode enzymes of benzylisoquinoline biosynthesis showed accumulate morphinan alkaloids. Investigations of two increased expression in P. somniferum varieties (Figure 2). cDNAs from this cluster led to the characterization of These included early pathway genes such as tyrosine salutaridine reductase leading to morphinan alkaloids and decarboxylase, 6OMT, and 4¢OMT, as well as genes involved
Figure 2. Gene expression analysis of Papaver species. Two clusters consisting of expressed sequence tags exhibiting increased expression in Papaver somniferum varieties are shown. The cDNAs coding for enzymes in benzylisoquinoline biosynthesis are indicated in blue and the cDNA characterized in this study is shown in red.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 Benzylisoquinoline O-methyltransferase 59 in the biosynthesis of specific benzylisoquinoline subclasses Gene expression analysis such as the morphinan alkaloid-specific salutaridinol-7-O- acetyltransferase and codeinone reductase 1, the laudanine- The transcript profiling revealed higher expression of specific 7OMT or the protoberberine-type specific berberine A21G11 in P. somniferum varieties compared to other bridge enzyme (Ziegler et al., 2006). The rationale behind Papaver species, although the expression level varied in that study was the isolation and characterization of novel individual plants of each variety. In only one variety (Papa; cDNAs involved in benzylisoquinoline biosynthesis based Figure 2) was A21G11 expression elevated in all individual on their expression profiles. This led to the identification of plants. All P. somniferum varieties produce morphinan hitherto uncharacterized cDNAs, for example the morphinan alkaloids (Ziegler et al., 2006). However, electrospray Fou- alkaloid-specific salutaridine reductase (Ziegler et al., 2006). rier transform ion cyclotron resonance mass spectrometry Another cDNA showing increased expression in P. som- (ESI-FT-ICRMS) showed the most prominent peaks for niferum varieties and which is clustered together with other masses indicative of other BIAs in some varieties. The most benzylisoquinoline biosynthetic genes was EST A21G11 abundant masses in the varieties Papa and Fool Ori were at (Figure 2). This EST with a length of 137 bp did not match to m/z 340.15441 and m/z 340.15409 [M+H]+, indicating an + any other sequence when submitted to the non-redundant elemental composition of [C20H22NO4] . A corresponding database on the NCBI server using either the BLASTX or low abundant mass signal was also present in Papaver BLASTN algorithms, even when an expectation value of 10 dubium, but was not detected in the other P. somniferum was used as threshold. In order to obtain sequence infor- varieties or species shown in Figure 2. Subsequent liquid mation for the whole cDNA, the Genome Walker kit was chromatography-tandem mass spectrometry (LC-MS/MS) applied using genomic DNA from P. somniferum with analyses identified the peak at m/z 340 ([M+H]+) as papav- primers designed according to the A21G11 sequence. erine in Papa, Fool Ori and P. dubium based on a compari- Sequence analysis of the resulting DNA fragment of son of retention time and fragmentation pattern compared 2287 bp revealed an open reading frame of 1071 bp distrib- with a papaverine standard. This alkaloid profile suggests uted over two exons of 777 bp and 294 bp, respectively, high A21G11 expression in plants with high papaverine which are interrupted by an intron of 735 bp. The 137-bp content. In order to confirm the expression data, quantita- sequence of EST A21G11 is located in the 3¢ untranslated tive RT-PCR analysis was performed with all benzyliso- region 85 bp downstream from the stop codon at positions quinoline OMTs. 1899 to 2036. The gene encodes a protein of 357 amino acids This expression analysis revealed that 4 ¢OMT, 6OMT, with a molecular mass of 39.7 kDa and an isoelectric point 7OMT and A21G11 are expressed at higher levels in stems of 4.98. compared with leaves, roots or seedlings (Figure 4). In The translated open reading frame exhibits several seedlings, 7OMT was expressed at a higher level than in motifs, which are typical for SAM-dependent class II type leaves or roots, whereas 6OMT and 4 ¢OMT expression was O-methyltransferase, including the catalytic histidine. Most higher in these tissues compared with seedlings. A21G11 of the residues which are involved in SAM binding in was expressed at similar levels in leaves, roots and seed- chalcone O-methyltransferase and isoflavone O-methyl- lings – levels which were very low compared with the transferase (Zubieta et al., 2001) are conserved in the expression levels observed in stems. Similar expression A21G11 sequence, as well as in other OMTs from benzyl- levels of 4 ¢OMT and 7OMT were shown (increase by 13 and isoquinoline biosynthesis (Figure 3a). Phylogenetic analysis 29%, respectively) in stems of the P. somniferum variety of several OMTs from plant secondary metabolism showed Papa, which contained the highest levels of papaverine, and that A21G11 forms a clade together with 6OMT from a variety without detectable levels of papaverine. While P. somniferum and C. japonica, which is well separated 6OMT and A21G11 seem to exhibit minor increases in from the 4¢OMTs (Figure 3b). A21G11 exhibited the highest expression, by 125 and 76%, respectively (Figure 4), there similarity to 6OMT from P. somniferum, followed by 6OMT was no statistical difference because of considerable from C. japonica (68 and 55% amino acid identity, respec- variation among samples. tively). The 4¢OMTs showed between 40 and 46% amino acid Overexpression and functional characterization of identity to A21G11. Distant from these enzymes, the recently recombinant A21G11 protein characterized 7OMTs from P. somniferum and E. californica form their own group. This is reflected by the lower The open reading frame was cloned into the expression percentage of identical amino acid residues (37%) between vector pQE30 coding for a six-histidine N-terminal extension A21G11 and both enzymes. This degree of sequence simi- and was over-expressed in Escherichia coli strain SG13009. larity was also observed for other OMTs of plant secondary The recombinant His-tagged protein had a relative molecular metabolism, which are not active in benzylisoquinoline mass of 40 kDa as determined by SDS–PAGE, which com- biosynthesis, such as caffeic acid and flavonoid O-meth- pared favorably with the calculated mass of 39.7 kDa de- yltransferases. duced from the translation of the cDNA. A native molecular
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(a)
(b)
Figure 3. Comparison of A21G11 to selected O-methyltransferases (OMTs) of plant secondary metabolism. (a) Amino acid sequence alignments of selected plant OMTs. The sequence alignment was performed using the CLUSTALW application (Thompson et al., 1994) of MegAlign (DNASTAR Inc., http://www.dnastar.com/). The protein sequence of A21G11 from Papaver somniferum (GenBank accession number FJ156103) was aligned to P. somniferum norcoclaurine 6-O-methyltransferase (Pso-6OMT), reticuline 7-O-methyltransferase (Pso-7OMT), 3¢-hydroxy N-methylcoclaurine 4¢-O- methyltransferase (Pso-4¢OMT) and Medicago sativa isoflavone 7-O-methyltransferase (Msa-I7OMT). Circles and box, residues involved in S-adenosyl-L-methionine (SAM) binding; triangle, catalytic histidine according to the Msa-I7OMT structure (PDB: 1FP2). (b) Neighbour joining phylogenetic tree of functionally characterized OMTs involved in plant secondary metabolism. The amino acid alignment was performed as described in (a). The tree was generated and visualized with the TREECON software (Yves van de Peer, University of Konstanz, Germany). Bootstrap values in per cent of 1000 trials are indicated. The source and accession numbers are: Eschscholzia californica Eca-4¢OMT (Inui et al., 2007); Pso-4¢OMT (AAP45314); Coptis japonica (Cja)-4¢OMT (Q9LEL5); Cja-6OMT (Q9LEL6); Pso-6OMT (AAQ01699); A21G11 (FJ156103); Hlu-OMT2, Humulus lupulus chalcone/xanthohumol OMT (ABZ89566); Rch-CafOMT, Rosa chinensis var spontanea caffeic acid OMT3 (BAC78828); Hlu-OMT1, H. lupulus desmethylxanthohumol OMT (ABZ89565); Eca-7OMT (BAE79723); Pso-7OMT (AAQ01668); Pta-AEOMT, Pinus taeda hydroxycinnamic acid/hydroxycinnamoyl-CoA ester OMT (AAC49708); Cja-SOMT, C. japonica scoulerine OMT (BAA06192); Msa-IL2¢OMT, Medicago sativa isoliquiritigenin 2¢OMT (AAB48059); TtuOMT1, Thalictrum tuberosum OMT1 (AAD29841); Cro-Caf3OMT, Catharanthus roseus caffeic acid 3OMT (Q8W013); Ath-F3¢OMT, Arabidopsis thaliana flavonol 3¢OMT (AAB96879); Msa-CafOMT, Medicago sativa caffeic acid OMT (P28002); Cbr-CafOMT, Clarkia breweri caffeic acid OMT (AAB71141); RchCafOMT, Rosa chinensis caffeic acid OMT (CAD29457); Cja-CoOMT, C. japonica columbamine OMT (BAC22084); Mpi-F8OMT, Mentha piperita flavonoid 8OMT (AAR09600); Rhy-OOMT, Rosa hybrid cultivar orcinol OMT (AAM23004); Rgr-3,5DMPOMT, Ruta graveolens 3,5-dimethoxyphenol OMT (AAX82431); Oba-CVOMT, Ocimum basilicum chavicol OMT (AAL30423); Cro-F4¢OMT, Catharanthus roseus flavonoid 4¢OMT (AAR02420); Msa-I7OMT (AAC49928). mass of 72 kDa was determined by gel filtration on a cali- Based on sequence homology to class II type OMTs of brated Superdex HL 200 column, suggesting the dimeric benzylisoquinoline metabolism, 16 alkaloids were tested as nature of the overexpressed protein (data not shown). possible substrates. Additionally, four phenolic compounds
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 Benzylisoquinoline O-methyltransferase 61
activity in the organic phase after extraction of the enzyme assays. Reticuline, the N-methylated derivative of norreticu- line, was not accepted as a substrate, nor were any tetrahydrobenzylisoquinolines containing methylated nitro- gens. Moreover, the enzyme seems to be dependent on the presence of a methoxy group at positions 6 and/or 4¢, since norlaudanosoline is not converted. Tetrahydrobenzyliso- quinolines with methoxy groups at position 7 and 3¢,asin norisoorientaline, are not tolerated as well. The BIAs of more complex structures such as scoulerine, or the promorphin- ans and morphinans salutaridinol, oripavine, codeine or morphine did not serve as substrates. The same was true for the tetrahydroisoquinoline salsolinol and the phenolic compounds. Figure 4. Gene expression analysis of benzylisoquinoline O-methyltransfe- Structural analysis of the reaction product rases (OMTs). The transcript levels of 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransfer- The HPLC analysis of the reaction products with norreticu- ase (4¢OMT), norcoclaurine 6-O-methyltransferase (6OMT), reticuline 7-O- methyltransferase (7OMT) and norreticuline 7-O-methyltransferase (N7OMT) line as the substrate showed a new peak eluting 3 min after in different organs of the high morphine producing variety (PaSo) and in the substrate peak, which could only be detected in incu- stems of a high papaverine producing variety (Papa) of P. somniferum were ) bations with active enzyme (Figure 6a). The LC-MS analysis analyzed. The relative transcript levels (2 DDCt) were calculated using elonga- + tion factor 1a as the normalizer and the DCt value of 4¢OMT in PaSo stems as revealed a [M+H] ion at m/z 330 for the product, indicating the calibrator. single methylation of norreticuline ([M+H]+, m/z 316; Fig- ure 6b). However, there are several positions which can be O-methylated, resulting in norlaudanine or norcodamine, (caffeic acid, vanillic acid, guaiacol and catechol) which are respectively. Additionally, N-methylation to reticuline can- known to be accepted by several class II type OMTs, as well not be ruled out. These three possibilities can be differenti- as the flavonoid quercetin and the coumarin esculetin were ated by their electrospray ionization (ESI) MS-fragmentation tested (Figure 5). Although homologous to 6OMT, norcocla- pattern. Tetrahydrobenzylisoquinoline alkaloids are cleaved urine did not serve as a substrate for A21G11, whereas into their isoquinoline and benzyl moieties. Norreticuline incubations with norreticuline exhibited substantial radio- shows a fragment at m/z 178, which represents the
Figure 5. Substrate specificity of A21G11. The structures of compounds tested as potential substrates of the Papaver somniferum O-meth- yltransferase A21G11 are displayed. Grey shad- ing denotes the converted compound.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 62 Silke Pienkny et al.
(a) isoquinoline part of the molecule (Figure 6; Schmidt et al., 2005). This fragment was shifted by 14 mass units towards higher masses in the reaction product, resulting in a corre- sponding signal at m/z 192. This suggests that methylation takes place at the isoquinoline moiety. In contrast, 3¢-O- methylation would have resulted in norcodamine and the mass of the isoquinoline fragment would have been unchanged. However, the ESI-time-of-flight (TOF) MS data did not show a corresponding fragment ion with a mass at m/z 178 (Figure 6b). Furthermore, the loss of ammonia leads to a fragment at m/z 299 for norreticuline. This fragment was also shifted by 14 mass units in the product, yielding a signal at m/z 313. An N-methylation would have led to a fragment at m/z 299 because of a loss of methylamine ([M+H– (b) + NH2CH3] ). Therefore, reticuline can be excluded as a pos- sible product. Based on this fragmentation pattern, and since position 6 is already methylated in norreticuline, it can be concluded that position 7 is methylated by the enzyme, yielding norlaudanine. Considering the substrate and product specificity of the A21G11 protein, it was named norreticuline 7-O-methyl- transferase (N7OMT). The recombinant protein exhibited its highest activity at 35 C, with half-maximum activity at 25 C and 40 C, respectively. The pH dependence revealed a broad optimum between pH 7.0 and pH 9.5, with strongly reduced
activity at pH 6.5 and pH 10.0. The enzyme exhibited a Km of (c) )1 44 Æ 5 lM (n = 6) and a kcat of 0.074 sec for norreticuline,
and a Km of 19 Æ 6 lM (n = 3) for the cofactor SAM. DISCUSSION Comparison of N7OMT with other OMTs in benzylisoquinoline biosynthesis O-Methylation is a common theme in the decoration of the benzylisoquinoline backbone, which leads to their structural diversity. Several benzylisoquinoline OMTs have been characterized, some of which exhibit distinct regio- and substrate specificities whereas some show a broader sub- strate range. Based on substrate specificity, N7OMT showed a very narrow substrate range with norreticuline as the only methyl acceptor (Figure 5). The contribution of the addi- tional N-terminal 6· histidine tag to this restrictive specificity was not investigated. The recombinant enzyme exhibited strong discrimination between N-methylated and N-deme- thylated benzylisoquinolines, as it is able to accept norreti- culine but not reticuline. Furthermore, all tested compounds Figure 6. Product identification of norreticuline conversion by A21G11 containing an N-methyl group are not converted. This is [=norreticuline 7-O-methyltransferase (N7OMT)]. unique among all cloned OMTs which are specific for simple (a) The HPLC chromatograms. Upper line, norreticuline substrate; middle line, after incubation with bacterial extracts without A21G11 protein; lower line, in tetrahydrobenzylisoquinolines. The 4¢OMTs from E. califor- the presence of A21G11 protein. nica, C. japonica, and P. somniferum accept laudanosoline (b) Electrospray ionization-time-of-flight (ESI-TOF) mass spectra of the as well as norlaudanosoline, although there is a preference substrate norreticuline at retention time (Rt) = 17.3 min on HPLC (top panel), and of the product at Rt = 20.0 min (lower panel). for the N-methylated substrate (Morishige et al., 2000; Zie- (c) Fragmentation patterns of norreticuline, and the possible enzyme products gler et al., 2005 Inui et al., 2007), whereas C. japonica 6OMT norlaudanine, norcodamine, and reticuline. exhibits equal activities for both compounds (Morishige
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 Benzylisoquinoline O-methyltransferase 63 et al., 2000). Papaver somniferum 6OMT accepts the specificity of N7OMT adds a new member to the range of N-demethylated norprotosinomenine but not protosinome- benzylisoquinoline OMTs. nine (Ounaroon et al., 2003). However, this enzyme converts Phylogenetic analysis revealed that N7OMT forms one the N-methylated isoorientaline, showing that modifications cluster together with 6OMTs (Figure 3b). Considering that at the heteroatom are tolerated for some compounds. Only other benzylisoquinoline OMTs are grouped according to 7OMT from P. somniferum also discriminates between their regiospecifities one could have expected a closer N-methylated and N-demethylated substrates. This enzyme relationship of N7OMT to 7OMTs. A similar pattern was displays exclusive preference for N-methylated tetrahyd- observed for OMTs capable of 6-O-methylation of tetrahyd- robenzylisoquinolines, since it is able to methylate reticuline robenzylisoquinolines. Here, the 6OMTs from C. japonica but not norreticuline. However, this enzyme also accepts and P. somniferum are not closely related to OMT1 from phenolic compounds and is not as specific for tetrahydrob- T. tuberosum, which also possesses norcoclaurine 6-O- enzylisoquinoline alkaloids as N7OMT (Ounaroon et al., methyltransferase activity. However, 7OMT from P. som- 2003). niferum as well as OMT1 from T. tuberosum accept phenolic It is found that N7OMT is not active with substrates in compounds with a higher catalytic efficiency than their which the methoxy and hydroxyl substitution pattern of the preferred tetrahydrobenzylisoquinoline substrate (Frick and tetrahydrobenzylisoquinoline backbone is different from Kutchan, 1999; Ounaroon et al., 2003). The promiscuous norreticuline. Even the non-methylated norlaudanosoline nature of 7OMT as well as OMT1 from T. tuberosum is not accepted as a substrate. This compound was methy- suggested that tetrahydrobenzylisoquinoline OMTs might lated by all other tetrahydrobenzylisoquinoline OMTs when have evolved from OMTs of the phenylpropanoid pathway it was tested as a substrate (Morishige et al., 2000; Ziegler (Frick and Kutchan, 1999; Ounaroon et al., 2003). Further et al., 2005; Inui et al., 2007). In contrast to N7OMT, 4¢OMT, speciation to 4¢OMT and 6OMT is believed to be the result of 6OMT, and 7OMT are more promiscuous with regard to the divergent evolution. Substrate specificity and sequence methylation pattern of the substrates. Although we used a homology suggested that 4¢OMTs have evolved further than wide range of compounds, we cannot exclude the possibility the 6OMTs (Ziegler et al., 2005). However, residual 6OMT that other benzylisoquinolines which have not been activity detected for C. japonica and E. californica 4¢OMTs included in this study are accepted by N7OMT. Nevertheless, led to the assumption that 6OMT might have evolved by compared with the other benzylisoquinoline OMTs studied speciation after duplication of a 4¢OMT gene (Inui et al., so far, N7OMT exhibits stronger substrate specificity. To our 2007). The close relationship of N7OMT to 6OMT suggests knowledge, there is only one OMT showing a similar degree that in P. somniferum a duplication of 6OMT and further of specificity. A 3¢OMT was purified and characterized from speciation led to the generation of N7OMT. In contrast to Argemone platyceras cell cultures and accepted only 6-O- that, the evolution of N7OMT through speciation after methylnorlaudanosoline out of a large range of substrates duplication of 7OMT seems unlikely. (Rueffer et al., 1983b). However, molecular data for this Participation of N7OMT in papaverine biosynthesis enzyme are not available. None of the tetrahydrobenzylisoquinoline-specific OMTs In the last decade considerable progress has been achieved cloned so far accepted norreticuline as a substrate in the cloning of cDNAs implicated in benzylisoquinoline (Morishige et al., 2000; Ounaroon et al., 2003; Ziegler et al., biosynthesis. All of the basic pathway genes up to reticuline 2005). Only Thalictrum tuberosum OMT2 (Frick and Kut- have been elucidated, as well as a major portion of the steps chan, 1999) showed some activity with norreticuline. leading to berberine and morphine (Ziegler and Facchini, However, this enzyme displayed a broad substrate range 2008). Considering the estimated number of 2500 benzyl- and is 10-fold more active towards caffeic acid and isoquinoline structures, many biosynthetic steps remain to catechol. Recombinant SOMT from C. japonica also be discovered, and current data on the biosynthetic sequence showed little side activity with norreticuline, with 10% of entire side pathways often reveal several options. conversion compared to the major substrate scoulerine The biogenic origin of papaverine is one of those exam- (Morishige et al., 2002). The regiospecificity has not been ples. Feeding experiments suggested reticuline and norori- determined for both enzymes. To our knowledge there is entaline as well as norreticuline to be possible pathway only one example where methylation of norreticuline could intermediates (Brochmann-Hanssen et al., 1971, 1975). After be measured in plant extracts. This activity was purified reticuline is converted by 7OMT (Ounaroon et al., 2003), from A. platyceras cell cultures and exhibited 1% activity laudanine might be methylated at the 3¢ position to laud- compared with the preferred substrate norlaudanosoline. anosine, which is N-demethylated to tetrahydropapaverine, This enzyme was shown to methylate norlaudanosoline at the immediate precursor of papaverine (Figure 7). The need positions 6 and 7 in a ratio of 4:1, but the regiospecificity for an N-demethylating step represents a major obstacle for for norreticuline was not determined (Rueffer et al., 1983a). support of a reticuline-based pathway since an enzyme Summarizing the available data, the substrate- and regio- catalyzing this reaction had not yet been detected in plants
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 64 Silke Pienkny et al.
Figure 7. Proposed scheme for papaverine biosynthesis in Papaver somniferum. Solid arrows denote conversions for which spe- cific enzymes have been cloned or characterized, or which can be performed in vitro by the indicated enzymes, respectively. For the conver- sions shown as broken arrows, an enzyme has not yet been detected in papaverine-accumulat- ing plants. 4¢OMT, (S)-3¢hydroxy N-methylcocla- urine 4¢O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; CYP80B3, (S)-N-methyl coclaurine 3¢hydroxylase; N7OMT, (S)-norreti- culine 7-O-methyltransferase.
with any benzylisoquinoline substrate. The incorporation of ruled out that N7OMT is involved in this pathway, especially labelled reticuline into papaverine was low compared with because of its preference for N-demethylated tetrahydrob- nororientaline or norreticuline (Brochmann-Hanssen et al., enzylisoquinolines. 1971). Additionally, feeding experiments did not suggest a The considerable activity of 4¢OMT towards 6-O-methyl- turnover of laudanine to papaverine, and laudanine accu- norlaudanosoline suggests a possible participation in a mulates in P. somniferum without detectable levels of norreticuline pathway (Morishige et al., 2000; Ziegler et al., papaverine. This indicates that methylation products of 2005). Together with the cloning of a norreticuline specific reticuline are not likely intermediates in papaverine biosyn- N7OMT, two enzymes able to catalyze O-methylation in a thesis (Brochmann-Hanssen et al., 1975; Schmidt et al., norreticuline based pathway have now been characterized 2007). from P. somniferum. However, an enzyme catalyzing the Considering N-demethylated tetrahydrobenzylisoquino- 3¢-O-methylation of norlaudanine is still missing. The 3¢OMT lines as intermediates, (S)-coclaurine could be diverted from A. platyceras has not been tested with norlaudanine towards papaverine biosynthesis by 3¢-hydroxylation to (Rueffer et al., 1983b) and the existence of a 3¢-OMT in 6-O-methylnorlaudanosoline. Further methylations might papaverine-producing plants has not been reported. occur, first at 3¢ followed by methylations at 4¢ and 7, when The feeding experiments, the properties of all available nororientaline represents an intermediate, or in the order 4¢, tetrahydrobenzylisoquinoline OMTs, and the slightly higher 7, and 3¢ in the case of a norreticuline based pathway expression of N7OMT in a papaverine-accumulating P. som- (Figure 7). In both pathways, a 3¢-hydroxylase is required for niferum variety, as observed from the macroarray analysis, conversion of coclaurine to 6-O-methylnorlaudanosoline. suggest that papaverine biosynthesis proceeds via norreti- The involvement of N-methylcoclaurine 3¢-hydroxylase culine. However, since higher N7OMT expression in papav- (CYP80B subfamily) can be ruled out, since it is strictly erine-accumulating plants cannot be clearly deduced from dependent on the presence of an N-methyl group (Pauli and the quantitative RT-PCR data, and because of the missing Kutchan, 1998; Huang and Kutchan, 2000). Loeffler and Zenk links in any of the possible pathways, transgenic approaches (1990) found an enzyme activity described as phenolase in are required to clearly resolve the biosynthesis of papaver- Berberis cell cultures converting coclaurine to 6-O-methyl- ine. Unfortunately, opium poppy transformation is a tedious norlaudanosoline. Subsequently, this compound might be and time-consuming task, but the availability of N7OMT methylated at the 3¢ position in a nororientaline pathway. cDNA provides a promising candidate for these experiments Such an activity has been detected in A. platyceras cell in order to elucidate papaverine biosynthesis. culture (Rueffer et al., 1983b). However, this enzyme and the phenolase have not yet been detected in papaverine- EXPERIMENTAL PROCEDURES producing plants. It is less likely that 4¢OMT and 7OMT are Plant material involved in that pathway: 4¢OMT exhibited no activity towards nororientaline and 7OMT is strictly dependent on The seeds of the Papaver plants were obtained either from the seed an N-methyl group, which excludes methylation of norcod- stock collection of the Department of Natural Product Biotechnol- ogy of the Leibniz Institute of Plant Biochemistry, Halle, Germany or amine (Ounaroon et al., 2003; Ziegler et al., 2005). Norcod- were the kind gift of Tasmanian Alkaloids Pty Ltd, Westbury, amine could not be tested as a substrate for N7OMT since Tasmania, Australia. Papaver somniferum L. plants were grown this compound was not available. Therefore, it cannot be outdoors in Saxony-Anhalt, Germany.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 Benzylisoquinoline O-methyltransferase 65
Analytical methods TAGTATCAGCGGGAGGG-3¢, A21G11-GW2: 5¢-GCAAGACGAAT- CACAGAATCAGATACGAAGCC-3¢, and AP1: 5¢-GTAATACGACT- The HPLC analysis was performed using an LC 1100 series Agilent CACTATAGGGC-3¢. The PCR fragments were cloned into pGEMT system (http://www.agilent.com/) equipped with a Lichrospher 60 (Promega, http://www.promega.com/), propagated in E. coli strain RP-select B column (250 · 4 mm, 5 lm; Merck, http://www.merck.- XL1BlueMRF and sequenced using the ABI Prism Big Dye Termi- com/) and a solvent system consisting of A, CH3CN–H2O (2:98; v/v); nator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). B, CH3CN–H2O (98:2; v/v) each with 0.1% (v/v) formic acid. The Sequencing reactions were run after removal of excess dye on the gradient was from 0% B to 46% B in 25 min with a hold for 5 min ABI Prism 3100 Sequencer (http://www.appliedbiosystems.com). followed by an increase to 100% B in 2 min and a hold for 3 min at a Genome Walking was performed twice until the sequence of the )1 flow rate of 1 ml min . The detection wavelength was set to gene’s 5¢ end could be detected. 282 nm. The entire open reading frame was obtained by RT-PCR from The ESI-MS measurements and LC separations were carried out P. somniferum stem mRNA using MLV Reverse Transcriptase (Pro- on a Mariner TOF mass spectrometer (Applied Biosystems, http:// mega) and PfuUltra Hotstart DNA Polymerase (Stratagene, http:// www3.appliedbiosystems.com/) equipped with a Turbo Ion Spray www.stratagene.com/) with the following primers containing BamHI source (PE-Sciex) using an LC1100 series Agilent system adapted to and PstI restriction sites: A21G11f2: 5¢-gaatggatccATGGAAGTAGT- )1 flow rates of 0.2 ml min . Samples were injected on a Supersphere TAGCCAGATTG-3¢ and A21G11r2: 5¢-gctactgcagTTAATAAACCTCA- 60 RP-Select B column (125 · 2 mm, 5 lm; Merck). The following LC ATTATAGATTG-3¢ using the following PCR conditions: five cycles of conditions were used: solvent A, CH3CN–H2O (2:98; v/v) and solvent 94 C for 30 sec, 45 C for 30 sec, 72 C for 2 min followed by 25 cycles B, CH3CN–H2O (98:2; v/v), 0.2% (v/v) formic acid in both solvents. of 94 C for 30 sec, 55 C for 30 sec, 72 C for 2 min and a final The gradient increased from 0% to 46% B in 25 min, to 90% in 1 min extension at 72 C for 5 min. After A-tailing with Taq-DNA polymer- and was held at 90% for 7 min; post time was 5 min. The TOF mass ase, the amplified PCR product was cloned into pCR2.1 (Invitrogen, spectrometer was operated in the positive ion mode, with nebulizer http://www.invitrogen.com/) and sequenced. For heterologous over- )1 )1 gas (N2) flow of 0.5 L min , curtain gas (N2) flow of 1.5 L min , and expression the plasmid was digested with endonucleases BamHI )1 heater gas flow of (N2) 7 L min . The spray tip potential of the ion and PstI (Fermentas), ligated into pQE32 (Qiagen, http://www. source was 5.5 kV, the heater and quadrupole temperature were qiagen.com/), and transformed in E. coli cells (strain SG13009). 360 C and 140 C, respectively, the nozzle potential was set to 200 V, For overexpression, the cells were induced with 1 mM isopropyl- and the detector voltage to 1.95 kV. The other settings varied b-D-thiogalactopyranoside, incubated at 4 C overnight, then har- depending on tuning. vested and sonicated in extraction buffer (50 mM potassium The positive ion ESI-collision-induced dissociation (CID) mass phosphate pH 7, 300 mM NaCl, 10% (v/v) glycerol, 1 mM spectra for the detection of papaverine in extracts from P. som- b-mercaptoethanol, 1% (v/v) Tween 20, 750 lgml)1 lysozyme). niferum var. Papa, P. somniferum var. FoolOri, and P. dubium After removal of the cell debris by centrifugation, the supernatant were obtained from a TSQ Quantum Ultra AM system (Thermo was loaded onto a cobalt affinity column (Talon; Clontech, http:// Scientific, http://www.thermo.com) equipped with a hot ESI source www.clontech.com/), followed by washing with extraction buffer (HESI; electrospray voltage 3.0 kV, sheath gas nitrogen; vaporizer without Tween 20 and lysozyme. The His-tagged O-methyltrans- temperature 50 C, capillary temperature 250 C). The MS system is ferase was eluted in washing buffer with an imidazole concentra- coupled to a Surveyor Plus micro-HPLC (Thermo Electron, http:// tion of 200 mM. The purified protein was desalted using PD10 www.thermo.com/), equipped with a RP18 column (5 lm, columns (Amersham Biosciences, http://www.gelifescience.com) 150 · 1 mm, Hypersil GOLD, Thermo Scientific). A gradient system in storage buffer [50 mM potassium phosphate pH 7.5, 20% (v/v) was used for separation, starting from H2O:CH3CN 85:15 (v/v) to glycerol]. The purity of the enzymes was checked by SDS–PAGE H2O:CH3CN 5:95 (v/v), each containing 0.2% (v/v) acetic acid, (12% polyacrylamide) according to Laemmli (1970). After purifica- within 15 min, followed by a hold for a further 30 min at a flow tion the protein concentration was determined with Bradford )1 rate of 50 ll min . The CID mass spectra of papaverine reagent at 595 nm (Bradford, 1976). (RT = 9.9 min) were recorded during the HPLC run with a collision energy of 30 eV (collision gas argon, collision gas pressure Enzyme assays 1.5 mTorr). The ESI-CID mass spectrum of papaverine is [m/z (relative The standard enzyme assay reaction mixture (80 ll) consisted of + intensity, %)]: 340 ([M+H] , 20), 324 (95), 296 (20), 202 (100), 187 50 mM potassium phosphate pH 7, 25 mM sodium ascorbate, 14 (8), 171 (34). The LC-MS/MS data were in excellent agreement with 250 lM S-adenosyl-L-methionine (SAM), [methyl- C]SAM (20 those of authentic papaverine. The fragmentation of papaverine is 000 c.p.m., 1.58 lM), 150 lM substrate, and 4.2 lg enzyme and was described elsewhere (Wickens et al., 2006). incubated for 20 min at 35 C. Substrates were obtained from the The high-resolution positive ion ESI mass spectra were obtained compound collection of the department or synthesized from buy- as described in Ziegler et al. (2006). able precursors. The enzymatic reaction was terminated by the addition of 100 llof1M NaHCO3 and products were extracted with Full-length cDNA cloning of N7OMT and protein expression 200 ll ethyl acetate in the case of benzylisoquinoline substrates. For in E. coli other substrates, the reaction was acidified by HCl prior to extrac- tion. The organic phase was added to 4 ml of scintillation cocktail For the generation of full-length cDNA for EST A21G11, the Genome Ultima GOLD MV (Perkin Elmer, http://www.perkinelmer.com/), and Walker Kit (BD Biosciences, http://www.bdbiosciences.com/) was the radioactivity was quantified with a Beckman Coulter LS 6500 used according to the manufacturer’s instructions. Genomic DNA liquid scintillation counter (http://www.beckman.com/). For product isolated from P. somniferum stems was used as a template. The identification on LC-MS, the enzyme assays were performed with- endonucleases DraI, EcoRV, PvuII and StuI were used to produce out the addition of radioactively labelled SAM. The influence of pH different libraries with genomic fragments. The PCR conditions with on enzyme activity was monitored in sodium citrate (pH 5.5–6.5), Long PCR Enzyme Mix (Fermentas, http://www.fermentas.com/) potassium phosphate (pH 6.0–8.4), 2-amino-2-(hydroxymethyl)-1,3- were as follows: 29 cycles of 96 C for 10 sec, 50 C for 5 sec, 60 C propanediol (TRIS)-HCl (pH 8.0–9.5) and glycine/NaOH (pH 9.0–10) for 4 min using the primers A21G11-GW1: 5¢-GATGCAAAGATG- buffered solutions.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 66 Silke Pienkny et al.
For enzyme kinetic analysis, the assays were performed under Brochmann-Hanssen, E., Leung, A.Y., Fu, C.C. and Zanati, G. (1971) Opium linear product formation conditions which were achieved by alkaloids X. Biosynthesis of 1-benzylisoquinolines. J. Pharm. Sci. 60, 1672– adjusting the protein concentration or the incubation time. The 1676. Brochmann-Hanssen, E., Chen, C.H., Chen, C.R., Chiang, H.C., Leung, A.Y. and substrate concentration ranged between 6.25 and 300 lM at a McMurtrey, K. (1975) Opium alkaloids. Part XVI. The biosynthesis of constant SAM concentration of 250 lM. The kinetic data for SAM 1-benzylisoquinolines in Papaver somniferum. Preferred and secondary were collected by varying the SAM concentration between 12.5 pathways; stereochemical aspects.J. Chem. Soc. [Perkin 1], 16, 1531–1537. and 250 lM at a constant norreticuline concentration of 150 lM. Chung, K.F. (2005) Drugs to suppress cough. Expert Opin. Investig. Drugs, 14, Although substrate inhibition was not evident up to 300 lM norret- 19–27. iculine, no further increase in the velocity could be observed above a Colombo, M.L. and Bosisio, E. (1996) Pharmacological activities of Chelido- concentration of 150 lM. Saturation curves and calculation of kinetic nium majus L (Papaveraceae). Pharmacol. Res. 33, 127–134. parameters were obtained according to Michaelis–Menten kinetics Frick, S. and Kutchan, T.M. (1999) Molecular cloning and functional expres- in Kaleidagraph (Synergy Software, http://www.synergy.com/). sion of O-methyltransferases common to isoquinoline alkaloid and phe- nylpropanoid biosynthesis. Plant J. 17, 329–339. Quantitative real-time PCR analysis Goodman, A.J., Le Bourdonnec, B. and Dolle, R.E. (2007) Mu opioid receptor antagonists: recent developments. Chem. Med. Chem. 2, 1552–1570. Total RNA was extracted from stems, seedlings, leaves, and roots of Huang, F.C. and Kutchan, T.M. (2000) Distribution of morphinan and P. somniferum var. Paso and from stems of P. somniferum var. benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum. Phytochemistry, 53, 555–564. Papa using TRIZOL reagent (Invitrogen). The RNA was treated with Inui, T., Tamura, K., Fujii, N., Morishige, T. and Sato, F. (2007) Overexpression RNase Inhibitor and DNaseI (Roche, http://www.roche.com/) of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the according to the manufacturer’s instructions. Total RNA was rate-limiting step in benzylisoquinoline alkaloid biosynthesis in cultured quantified via absorption at 260 nm and the quality was checked by Eschscholzia californica. Plant Cell Physiol. 48, 252–262. gel electrophoresis. First-strand cDNA synthesis was performed Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of using 2 lg total RNA and Superscript II reverse transcriptase (Invi- the head of bacteriophage T4. Nature, 227, 680–685. trogen) according to the manufacturer’s protocol. The PCR ampli- Loeffler, S. and Zenk, M.H. (1990) The hydroxylation step in the biosynthetic fications were performed with the following oligonucleotides which pathway leading from norcoclaurine to reticuline. Phytochemistry, 29, were designed using the Primer Express software (Applied Bio- 3499–3503. Morishige, T., Tsujita, T., Yamada, Y. and Sato, F. (2000) Molecular charac- systems): 6OMT, 5¢-AACACTGGTGGAAAAGAGAGAACC-3¢ and terization of the S-adenosyl-L-methionine : 3 ‘-hydroxy-N-methylcoclaurine 5¢-CCTCAATTACAGATTGGACAGCAG-3¢;4¢OMT, 5¢-AGAGAGAG- 4‘-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in AACTGCAGAGGATTGG-3¢ and 5¢-CTTCAATGACAGACTGAATA- Coptis japonica. J. Biol. Chem. 275, 23398–23405. GCGC-3¢; 7OMT, 5¢-CTGATGATGGCACATACTACAGCTG-3¢ and Morishige, T., Dubouzet, E., Choi, K.B., Yazaki, K. and Sato, F. (2002) Molec- 5¢-GGAAATGCCGGAGTTCGAAT-3¢; N7OMT, 5¢-GATGCAGCAG- ular cloning of columbamine O-methyltransferase from cultured Coptis GTTTTGCTAGTTG-3¢ and 5¢-AGCTAACAAAGTCTCGCCCTCC-3¢; ef1 japonica cells. Eur. J. Biochem. 269, 5659–5667. (elongation factor 1a), 5¢-AGATGATTCCAACCAAGCCCA-3¢ and Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. and Kutchan, T.M. (2003) 5¢-CCTTGATGACACCAACAGCAACT-3¢. Each reaction contained a (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-meth- 20-ng RNA equivalent of cDNA, 1 pmol of gene-specific primers, yltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant and 5 ll SYBR Green PCR Master Mix (Applied Biosystems). The J. 36, 808–819. PCR reaction was performed in a MX 3000P Cycler (Stratagene) Pauli, H.H. and Kutchan, T.M. (1998) Molecular cloning and functional using the following protocol: 95 C for 10 min, 40 cycles 95 C for heterologous expression of two alleles encoding (S)-N-methylcoclaurine 30 sec, 60 C for 1 min, 72 C for 30 sec. After each PCR a melting 3’-hydroxylase (Cyp80b1), a new methyl jasmonate-inducible cytochrome curve was measured by heating the samples to 95 C for 1 min, P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosyn- followed by 60 C for 30 sec and a temperature increase to 95 C. thesis. Plant J. 13, 793–801. Control reactions were run with untranscribed RNA. All measure- Rueffer, M., Nagakura, N. and Zenk, M.H. (1983a) Partial purification and ments were performed with two technical and two biological rep- properties of S-adenosylmethionine (R),(S)-norlaudanosoline-6-O-methyl- licates. For each tissue, the delta of the threshold cycle (DC ) values transferase from Argemone platyceras cell cultures. Planta Med. 49, 131– t 137. for each gene were obtained by subtraction of the arithmetic mean Rueffer, M., Nagakura, N. and Zenk, M.H. (1983b) A highly specific O-meth- Ct values of the normalizing ef1 from the arithmetic mean Ct values yltransferase for nororientaline synthesis isolated from Argemone platyc- of each gene. The DDCt was calculated using the DCt value of 4¢OMT eras cell cultures. Planta Med. 49, 196–198. in PaSo stems as the calibrator. The Ct values of the normalizer ef1 Sato, Y., He, J.X., Nagai, H., Tani, T. and Akao, T. (2007) Isoliquiritigenin, one were 18.45 Æ 0.22 for PaSo stems, 17.14 Æ 0.01 for leaves, 17.25 Æ of the antispasmodic principles of Glycyrrhiza ularensis roots, acts in the 0.01 for roots, 16.56 Æ 0.52 for seedlings, and 17.06 Æ 0.75 for Papa lower part of intestine. Biol. Pharm. Bull. 30, 145–149. stems. Schmidt, J., Raith, K., Boettcher, C. and Zenk, M.H. (2005) Analysis of ben- zylisoquinoline type alkaloids by electrospray tandem mass spectrometry ACKNOWLEDGEMENTS and atmospheric pressure photoionization. Eur. J. Mass Spectrom. 11, 325– 334. Funding was provided by Deutsche Forschungsgemeinschaft, Bonn Schmidt, J., Boettcher, C., Kuhnt, C., Kutchan, T.M. and Zenk, M.H. (2007) (SPP1152, Priority Program ‘Evolution of Metabolic Diversity’). Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding. REFERENCES Phytochemistry, 68, 189–202. Shulgin, A.T. and Perry, W.E. (2002) The Simple Plant Isoquinolines. Berkeley, Boswell-Smith, V., Spina, D. and Page, C.P. (2006) Phosphodiesterase inhib- CA: Transform Press. itors. Br. J. Pharmacol. 147, Suppl 1, S252–S257. Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y. and Sato, F. Bradford, M.M. 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Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improv- somniferum and eight morphine free Papaver species identifies an O- ing the sensitivity of progressive multiple sequence alignment through methyltransferase involved in benzylisoquinoline biosynthesis. Planta, sequence weighting, position-specific gap penalties and weight matrix 222, 458–471. choice. Nucleic Acids Res. 22, 4673–4680. Ziegler, J., Voigtla¨ nder, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, Wickens, J.R., Sleeman, R. and Keely, B.J. (2006) Atmospheric pressure C., Gesell, A. and Kutchan, T.M. (2006) Comparative transcript and ionisation mass spectrometric fragmentation pathways of noscapine and alkaloid profiling in Papaver species identifies a short chain dehydro- papaverine revealed by multistage mass spectrometry and in-source deu- genase/reductase involved in morphine biosynthesis. Plant J. 48, 177– terium labeling. Rapid Commun. Mass Spectrom. 20, 473–480. 192. Ziegler, J. and Facchini, P.J. (2008) Alkaloid biosynthesis: metabolism and Zubieta, C., He, X.Z., Dixon, R.A. and Noel, J.P. (2001) Structures of two nat- trafficking. Annu. Rev. Plant. Biol. 59, 735–769. ural product methyltransferases reveal the basis for substrate specificity in Ziegler, J., Diaz-Cha´ vez, M.L., Kramell, R., Ammer, C. and Kutchan, T.M. plant O-methyltransferases. Nat. Struct. Biol. 8, 271–279. (2005) Comparative macroarray analysis of morphine containing Papaver
Accession numbers for sequence data: Sequence data from this article have been deposited with the EMBL/GenBank data libraries under the accession numbers FJ156103 for P. somniferum norreticuline 7-O-methyltransferase (N7OMT).
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67