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 56 ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd Benzylisoquinoline O-methyltransferase 57 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 ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67 58 Silke Pienkny et al. 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