3′O-Methyltransferase, Ps3OMT, from opium poppy: involvement in papaverine biosynthesis

  • Parul Agarwal
  • Sumya Pathak
  • Ravi Shankar Kumar
  • Yogeshwar Vikram Dhar
  • Ashutosh Pandey
  • Sudhir Shukla
  • Prabodh Kumar TrivediEmail author
Original Article


Key message

Using, in silico, in vitro and in planta functional assays, we demonstrate that Ps3′OMT, an 3′-O methyl transferase is linked to papaverine biosynthesis in opium poppy.


Papaverine, one of the benzylisoquinoline alkaloids (BIA) synthesized in the medicinally important plant, Papaver somniferum, is known for the potent pharmacological properties. Papaverine biosynthesis has remained debatable as two different pathways, NH (involving N-desmethylated intermediates) and the NCH3 (involving N-methylated intermediates), have been proposed. In addition, there are several intermediate steps in both the proposed pathways that are not very well characterized in terms of specific enzymes. In this study, we report the identification and functional characterization of 3′O-methyltransferase (Ps3′OMT) which might participate in the 3′O-methylation of the intermediates in the papaverine biosynthesis. Comparison of transcript and metabolite profiles of high and low papaverine producing cultivar revealed the occurrence of a 3′O-methyltransferase, Ps3′OMT, which was abundant in aerial organs and shared 72% identity with the GfLOMT7 predicted to have 3′OMT activity. In silico studies based on homology modeling, docking and MD simulations predicted (S)-norlaudanine as the potential substrate forming a stable complex with Ps3′OMT. Suppression of Ps3′OMT through virus-induced gene silencing resulted in a remarkable decrease in the level of papaverine in comparison to control plants. The characterization of the functionally unique Ps3′OMT involved in BIA metabolism suggests an involvement of the NH pathway leading to papaverine biosynthesis.


Benzylisoquinoline alkaloids NH pathway O-Methyltransferase Opium poppy Papaver somniferum Papaverine biosynthesis 



Authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi for financial support under Network project (BSC-107). PA, SP as well as RK acknowledge Department of Biotechnology, Government of India and University Grants Commission (UGC) for Senior and Junior Research Fellowships. Authors acknowledge Dr. Surendra Pratap Singh, CSIR-NBRI for help in confocal microscopy.

Author contribution statement

PA, SP, SS and PKT designed the research; PA, RK and AP performed the experiments; YVD carried out the bioinformatics analysis; PA and PKT wrote the paper.

Compliance with ethical standards

Conflict of interest

Authors declare that they have no competing interests. All authors have read and approved the final manuscript.

Supplementary material

299_2019_2439_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1837 kb)


  1. Afzali M, Ghaeli P, Khanavi M, Parsa M, Montazeri H, Ghahremani MH, Ostad S (2015) Non-addictive opium alkaloids selectively induce apoptosis in cancer cells compared to normal cells. DARU J Pharm Sci 23:16CrossRefGoogle Scholar
  2. Agarwal P, Pathak S, Lakhwani D, Gupta P, Asif MH, Trivedi PK (2016) Comparative analysis of transcription factor gene families from Papaver somniferum: identification of regulatory factors involved in benzylisoquinoline alkaloid biosynthesis. Protoplasma 253:857–871CrossRefGoogle Scholar
  3. Agarwal AV, Gupta P, Singh D, Dhar YV, Chandra D, Trivedi PK (2017) Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression. Funct Integr Genom 17:477–490CrossRefGoogle Scholar
  4. Beaudoin GA, Facchini PJ (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240:19–32CrossRefGoogle Scholar
  5. Bella AJ, Brock GB (2004) Intracavernous pharmacotherapy for erectile dysfunction. Endocrine 23:149–155CrossRefGoogle Scholar
  6. Borchardt RT (1974) A rapid spectrophotometric assay for catechol-O-methyltransferase. Anal Biochem 58:382–389CrossRefGoogle Scholar
  7. Chang L, Hagel JM, Facchini PJ (2015) Isolation and characterization of O-methyltransferases involved in the biosynthesis of glaucine in Glaucium flavum. Plant Physiol 169:1127–1140CrossRefGoogle Scholar
  8. Charlier R (2013) Coronary vasodilators: international series of monographs on pure and applied biology division. In: Alexander P, Bacq ZM (eds) Modern trends in physiological sciences. Elsevier Science, New York, pp 76–78Google Scholar
  9. Dang TTT, Facchini PJ (2012) Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy. Plant Physiol 159:618–631CrossRefGoogle Scholar
  10. Dang TTT, Onoyovwe A, Farrow SC, Facchini PJ (2012) Biochemical genomics for gene discovery in benzylisoquinoline alkaloid biosynthesis in opium poppy and related species. Methods Enzymol 515:231–266CrossRefGoogle Scholar
  11. Dang TTT, Chen X, Facchini PJ (2015) Acetylation serves as a protective group in noscapine biosynthesis in opium poppy. Nat Chem Biol 11:104–106CrossRefGoogle Scholar
  12. Dastmalchi M, Park MR, Morris JS, Facchini PJ (2017) Family portraits: the enzymes behind benzylisoquinoline alkaloid diversity. Phytochem Rev 17:249–277CrossRefGoogle Scholar
  13. Desgagné-Penix I, Facchini PJ (2012) Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. Plant J 72:331–344CrossRefGoogle Scholar
  14. Dziggel C, Schäfer H, Wink M (2017) Tools of pathway reconstruction and production of economically relevant plant secondary metabolites in recombinant microorganisms. Biotechnol J 12:1600145CrossRefGoogle Scholar
  15. Farrow SC, Facchini PJ (2015) Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe2+-dependent dioxygenase from opium poppy. FEBS Lett 589:2701–2706CrossRefGoogle Scholar
  16. Farrow SC, Hagel JM, Beaudoin GA, Burns DC, Facchini PJ (2015) Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat Chem Biol 11:728–732CrossRefGoogle Scholar
  17. Galanie S, Thodey K, Trenchard IJ, Interrante MF, Smolke CD (2015) Complete biosynthesis of opioids in yeast. Science 349:1095–1100CrossRefGoogle Scholar
  18. Gurkok T, Turktas M, Parmaksiz I, Unver T (2015) Transcriptome profiling of alkaloid biosynthesis in elicitor induced opium poppy. Plant Mol Biol Report 33:673–688CrossRefGoogle Scholar
  19. Hagel JM, Facchini PJ (2010) Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat Chem Biol 6:273–275CrossRefGoogle Scholar
  20. Hagel JM, Facchini PJ (2012) Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells. Vitro Cell Dev Biol Plant 48:233–240CrossRefGoogle Scholar
  21. Hagel JM, Facchini PJ (2013) Biochemical genomics to investigate benzylisoquinoline alkaloid biosynthesis in opium poppy and related plants. Plant Cell Physiol 54:647–672CrossRefGoogle Scholar
  22. Han X, Lamshöft M, Grobe N, Ren X, Fist AJ, Kutchan TM, Spiteller M et al (2010) The biosynthesis of papaverine proceeds via (S)-reticuline. Phytochemistry 71:1305–1312CrossRefGoogle Scholar
  23. Joshi CP, Chiang VL (1998) Conserved sequence motifs in plant S-adenosyl-l-methionine-dependent methyltransferases. Plant Mol Biol 37:663–674CrossRefGoogle Scholar
  24. Khanna KR, Shukla S (1991) Inheritance of papaverine in Papaver somniferum L. and a morphological marker for high papaverine plants. Herba Hung 30:7–10Google Scholar
  25. Lam KC, Ibrahim RK, Behdad B, Dayanandan S (2007) Structure, function, and evolution of plant O-methyltransferases. Genome 50:1001–1013CrossRefGoogle Scholar
  26. Liu JK, Couldwell WT (2005) Intra-arterial papaverine infusions for the treatment of cerebral vasospasm induced by aneurysmal subarachnoid hemorrhage. Neurocrit Care 2:124–132CrossRefGoogle Scholar
  27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2−∆∆CT methods. Methods 25:402–408CrossRefGoogle Scholar
  28. Louie GV, Bowman ME, Tu Y, Mouradov A, Spangenberg G, Noel JP (2010) Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference. Plant Cell 22:4114–4127CrossRefGoogle Scholar
  29. Mishra S, Tripathi V, Singh S, Shanke K, Shukla R (2013) Wound induced transcriptional regulation of benzylisoquinoline pathway and characterization of wound inducible PsWRKY transcription factor from P. somniferum. PLoS One 8:e52784CrossRefGoogle Scholar
  30. Morishige T, Tsujita T, Yamada Y, Sato F (2000) Molecular characterization of the S-adenosyl-l-methionine: 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J Biol Chem 275:23398–23405CrossRefGoogle Scholar
  31. Morishige T, Dubouzet E, Choi KB, Yazaki K, Sato F (2002) Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells. Eur J Biochem 269:5659–5667CrossRefGoogle Scholar
  32. Narcross L, Fossati E, Bourgeois L, Dueber JE, Martin VJJ (2016) Microbial factories for the production of benzylisoquinoline alkaloids. Trends Biotechnol 34:228–241CrossRefGoogle Scholar
  33. Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H (1999) Non invasive quantitative detection and applications of non-toxic, S65T type green fluorescent protein in living plants. Plant J 18:455–463CrossRefGoogle Scholar
  34. Ounaroon A, Decker G, Schmidt J, Lottspeich F, Kutchan TM (2003) (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum—cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J 36:808–819CrossRefGoogle Scholar
  35. Pathak S, Lakhwani D, Gupta P, Mishra BK, Shukla S, Asif MH, Trivedi PK (2013) Comparative transcriptome analysis using high papaverine mutant of P. somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis. PLoS One 8:e65622CrossRefGoogle Scholar
  36. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 2513:1605–1612CrossRefGoogle Scholar
  37. Pienkny S, Brandt W, Schmidt J, Kramell R, Ziegler J (2009) Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L.). Plant J 60:56–67CrossRefGoogle Scholar
  38. Samanani N, Alcantara J, Bourgault R, Zulak KG, Facchini PJ (2006) The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy. Plant J 47:547–563CrossRefGoogle Scholar
  39. Sillanpää M, Koponen M (1978) Papaverine in the prophylaxis of migraine and other vascular headache in children. Acta Paediatr Scand 67:209–212CrossRefGoogle Scholar
  40. Stadler R, Zenk MH (1990) A revision of the generally accepted pathway for the biosynthesis of the benzyltetrahydroisoquinoline reticuline. Liebigs Ann Chem 1990:555–562CrossRefGoogle Scholar
  41. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  42. Thodey K, Galanie S, Smolke CD (2014) A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat Chem Biol 10:837–844CrossRefGoogle Scholar
  43. Uprety H, Bhakuni DS, Kapil RS (1975) Biosynthesis of papaverine. Phytochemistry 14:1535–1537CrossRefGoogle Scholar
  44. Wijekoon C, Facchini PJ (2012) Systematic knockdown of morphine biosynthesis in opium poppy using virus-induced gene silencing. Plant J 69:1052–1063CrossRefGoogle Scholar
  45. Winzer T, Kern M, King AJ, Larson TR, Teodor RI, Donninger SL, Li Y, Dowle AA, Cartwright J, Bates R, Ashford D (2015) Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein. Science 349:309–312CrossRefGoogle Scholar
  46. Zhao Y, Wang N, Zeng Z, Xu S, Huang C, Wang W, Liu T, Luo J, Kong L (2016) Cloning, functional characterization, and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn. Front Plant Sci 7:722Google Scholar
  47. Ziegler J, Diaz-Chávez ML, Kramell R, Ammer C, Kutchan TM (2005) Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta 222:458–471CrossRefGoogle Scholar
  48. Ziegler J, Voigtländer S, Schmidt J, Kramell R, Miersch O, Ammer C, Gesell A, Kutchan TM (2006) Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J 48:177–192CrossRefGoogle Scholar
  49. Zubieta C, Kota P, Ferrer JL, Dixon RA, Noel JP (2002) Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase. Plant Cell 14:1265–1277CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR-NBRI)LucknowIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)GhaziabadIndia
  3. 3.Department of BiochemistryUniversity of LucknowLucknowIndia

Personalised recommendations