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Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy

Nature Chemical Biology volume 11, pages 728–732 (2015)Cite this article

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Abstract

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

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Figure 1: Proposed two-step stereochemical inversion of (S)-reticuline to (R)-reticuline catalyzed by 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR) in opium poppy.
Figure 2: Maps of cDNAs and genes encoding reticuline epimerase (REPI), 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR).
Figure 3: Catalytic functions of PsREPI, PsDRS and PrDRS using (S)-reticuline, 1,2-dehydroreticuline and (R)-reticuline as substrates.
Figure 4: Virus-induced gene silencing in opium poppy supports the role of PsREPI in morphinan alkaloid biosynthesis.

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References

  1. Quasdorf, K.W. & Overman, L.E. Catalytic enantioselective synthesis of quaternary carbon stereocentres. Nature 516, 181–191 (2014).

    Article  CAS  Google Scholar 

  2. Hagel, J.M. & Facchini, P.J. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672 (2013).

    Article  CAS  Google Scholar 

  3. Gulland, J.M. & Robinson, R. Constitution of codeine and thebaine. Mem. Proc. Manchester Lit. Phil. Soc. 69, 79–86 (1925).

    CAS  Google Scholar 

  4. Kirby, G.W. Biosynthesis of the morphine alkaloids. Science 155, 170–173 (1967).

    Article  CAS  Google Scholar 

  5. Battersby, A.R., Foulkes, D.M. & Binks, R. Biosynthesis of morphine alkaloids. J. Chem. Soc. 33, 3323–3332 (1965).

    Article  CAS  Google Scholar 

  6. Hirata, K., Poeaknapo, C., Schmidt, J. & Zenk, M.H. 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 65, 1039–1046 (2004).

    Article  CAS  Google Scholar 

  7. De-Eknamkul, W. & Zenk, M.H. Purification and properties of 1,2-dehydroreticuline reductase from Papaver somniferum seedlings. Phytochemistry 31, 813–821 (1992).

    Article  CAS  Google Scholar 

  8. Allen, R.S. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 1559–1566 (2004).

    Article  CAS  Google Scholar 

  9. Facchini, P.J. & St-Pierre, B. Synthesis and trafficking of alkaloid biosynthetic enzymes. Curr. Opin. Plant Biol. 8, 657–666 (2005).

    Article  CAS  Google Scholar 

  10. Jirschitzka, J. et al. Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae. Proc. Natl. Acad. Sci. USA 109, 10304–10309 (2012).

    Article  CAS  Google Scholar 

  11. Ballance, G.M. & Dixon, R.A. Medicago sativa cDNAs encoding chalcone reductase. Plant Physiol. 107, 1027–1028 (1995).

    Article  CAS  Google Scholar 

  12. Desgagné-Penix, I., Farrow, S.C., Cram, D., Nowak, J. & Facchini, P.J. Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy. Plant Mol. Biol. 79, 295–313 (2012).

    Article  Google Scholar 

  13. Amann, M. & Zenk, M.H. Preparation of dehydrobenzylisoquinolines by immobilized (S)-tetrahydroprotoberberine oxidase from plant cell cultures. Phytochemistry 26, 3235–3240 (1987).

    Article  CAS  Google Scholar 

  14. Amann, M., Nagakura, N. & Zenk, M.H. Purification and properties of (S)-tetrahydroprotoberberine oxidase from suspension-cultured cells of Berberis wilsoniae. Eur. J. Biochem. 175, 17–25 (1988).

    Article  CAS  Google Scholar 

  15. Takemura, T., Ikezawa, N., Iwasa, K. & Sato, T. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells. Phytochemistry 91, 100–108 (2013).

    Article  CAS  Google Scholar 

  16. Beaudoin, G.A.W. & Facchini, P.J. Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis. Biochem. Biophys. Res. Commun. 431, 597–603 (2013).

    Article  CAS  Google Scholar 

  17. Dang, T.T.T. & Facchini, P.J. CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy. J. Biol. Chem. 289, 2013–2026 (2014).

    Article  CAS  Google Scholar 

  18. Dang, T.T.T., Chen, X. & Facchini, P.J. Acetylation serves as a protective functional group in noscapine biosynthesis in opium poppy. Nat. Chem. Biol. 11, 104–106 (2015).

    Article  CAS  Google Scholar 

  19. Morikawa, T. et al. Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato. Plant Cell 18, 1008–1022 (2006).

    Article  CAS  Google Scholar 

  20. Field, B. & Osbourn, A.E. Metabolic diversification—independent assembly of operon-like gene clusters in different plants. Science 320, 543–547 (2008).

    Article  CAS  Google Scholar 

  21. Martens, S. & Mithöfer, A. Flavones and flavone synthases. Phytochemistry 66, 2399–2407 (2005).

    Article  CAS  Google Scholar 

  22. Mizutani, M. & Sato, F. Unusual P450 reactions in plant secondary metabolism. Arch. Biochem. Biophys. 507, 194–203 (2011).

    Article  CAS  Google Scholar 

  23. Akashi, T. et al. Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones. Plant Cell Physiol. 40, 1182–1186 (1999).

    Article  CAS  Google Scholar 

  24. He, X.S. 1,2-Dehydroreticuline: conversion of iminium salts into enamimes. J. Nat. Prod. 56, 973–975 (1993).

    Article  CAS  Google Scholar 

  25. Battersby, A.R., Foulkes, D.M., Hirst, M., Parry, G.V. & Staunton, J. Alkaloid biosynthesis. Part XI. Studies related to the formation and oxidation of reticuline in morphine biosynthesis. J. Chem. Soc. C 1968, 210–216 (1968).

    Article  Google Scholar 

  26. Penning, T.M. The aldo-keto reductases (AKRs): overview. Chem. Biol. Interact. 234, 236–246 (2015).

    Article  CAS  Google Scholar 

  27. Wang, Y., Yi, H., Wang, M., Yu, O. & Jez, J.M. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase:stilbene synthase. J. Am. Chem. Soc. 133, 20684–20687 (2011).

    Article  CAS  Google Scholar 

  28. Brodelius, M., Lundgren, A., Mercke, P. & Brodelius, P.E. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum. Eur. J. Biochem. 269, 3570–3577 (2002).

    Article  CAS  Google Scholar 

  29. Sharma, H., Landau, M.J., Vargo, M.A., Spasov, K.A. & Anderson, K.S. First three-dimensional structure of Toxoplasma gondii thymidylate synthase-dihydrofolate reductase: insights for catalysis, interdomain interactions, and substrate channeling. Biochemistry 52, 7305–7317 (2013).

    Article  CAS  Google Scholar 

  30. Peters, R.J., Carter, O.A., Zhang, Y., Matthews, B.W. & Croteau, R.B. Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations. Biochemistry 42, 2700–2707 (2003).

    Article  CAS  Google Scholar 

  31. Elleuche, S. Bringing functions together with fusion enzymes—from nature's inventions to biotechnological applications. Appl. Microbiol. Biotechnol. 99, 1545–1556 (2015).

    Article  CAS  Google Scholar 

  32. Marienhagen, J. & Bott, M. Metabolic engineering of microorganisms for the synthesis of plant natural products. J. Biotechnol. 163, 166–178 (2013).

    Article  CAS  Google Scholar 

  33. Pillai, B. et al. Structural insights into stereochemical inversion by diaminopimelate epimerase: an antimicrobial drug target. Proc. Natl. Acad. Sci. USA 103, 8668–8673 (2006).

    Article  CAS  Google Scholar 

  34. Morrison, J.P., Read, J.A., Coleman, W.G. & Tanner, M.E. Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism. Biochemistry 44, 5907–5915 (2005).

    Article  CAS  Google Scholar 

  35. Marbaix, A.Y. et al. Extremely conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide repair. J. Biol. Chem. 286, 41246–41252 (2011).

    Article  CAS  Google Scholar 

  36. Stapon, A., Li, R. & Townsend, C.A. Synthesis of (3S,5R)-carbapenam-3-carboxylic acid and its role in carbapenem biosynthesis and the stereoinversion problem. J. Am. Chem. Soc. 125, 15746–15747 (2003).

    Article  CAS  Google Scholar 

  37. Suhre, K. Inference of gene function based on gene fusion events: the rosetta-stone method. Methods Mol. Biol. 396, 31–41 (2007).

    Article  CAS  Google Scholar 

  38. Nützmann, H.-W. & Osbourn, A. Gene clustering in plant specialized metabolism. Curr. Opin. Biotechnol. 26, 91–99 (2014).

    Article  Google Scholar 

  39. Winzer, T. et al. A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science 336, 1704–1708 (2012).

    Article  CAS  Google Scholar 

  40. Schiff, P.L. Bisbenzylisoquinoline alkaloids. J. Nat. Prod. 60, 934–953 (1997).

    Article  CAS  Google Scholar 

  41. Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014).

    Article  Google Scholar 

  42. Morishige, T., Tsujita, T., Yamada, Y. & Sato, F. 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, 23398–23405 (2000).

    Article  CAS  Google Scholar 

  43. Choi, K.B., Morishige, T., Shitan, N., Yazaki, K. & Sato, F. Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem. 277, 830–835 (2002).

    Article  CAS  Google Scholar 

  44. Ro, D.K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    Article  CAS  Google Scholar 

  45. Pompon, D., Louerat, B., Bronine, A. & Urban, P. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64 (1996).

    Article  CAS  Google Scholar 

  46. Dang, T.T.T. & Facchini, P.J. Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy. FEBS Lett. 588, 198–204 (2014).

    Article  CAS  Google Scholar 

  47. Liu, Y., Schiff, M. & Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002).

    Article  CAS  Google Scholar 

  48. Dinesh-Kumar, S.P., Anandalakshmi, R., Marathe, R., Schiff, M. & Liu, Y. Virus-induced gene silencing. Methods Mol. Biol. 236, 287–294 (2003).

    CAS  PubMed  Google Scholar 

  49. Desgagné-Penix, I. & Facchini, P.J. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. Plant J. 72, 331–344 (2012).

    Article  Google Scholar 

  50. Farrow, S.C. & Facchini, P.J. Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy. J. Biol. Chem. 288, 28997–29012 (2013).

    Article  CAS  Google Scholar 

  51. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Nelson (University of Tennessee) for assigning the CYP nomenclature. This work was supported by grants from Genome Canada, Genome Alberta, the Government of Alberta, the Canada Foundation for Innovation and the Natural Sciences and Engineering Research Council of Canada to P.J.F. S.C.F. and G.A.W.B. were recipients of scholarships from the Natural Sciences and Engineering Research Council of Canada. S.C.F. also received a scholarship from Alberta Innovates Technology Futures.

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  1. Guillaume A W Beaudoin

    Present address: Current address: Horticultural Sciences Department, University of Florida, Gainesville, Florida, USA.,

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  1. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

    Scott C Farrow, Jillian M Hagel, Guillaume A W Beaudoin & Peter J Facchini

  2. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada

    Darcy C Burns

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Contributions

S.C.F. performed all recombinant enzyme assays, virus-induced gene silencing experiments and mass spectrometric analyses, and co-wrote the manuscript. J.M.H. constructed the yeast expression vectors, performed all qRT-PCR experiments, and co-wrote the manuscript. D.C.B. conducted and interpreted the NMR analysis. G.A.W.B. contributed to the initial gene isolations. P.J.F. directed the research, prepared the figures and tables, and edited the manuscript.

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Correspondence to Peter J Facchini.

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P.J.F., S.C.F. and G.A.W.B. have filed a patent application (Patent Cooperation Treaty Application PCT/CA2014/0511164) covering compositions and methods related to the manufacture or (R)-reticuline and precursors thereof.

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Farrow, S., Hagel, J., Beaudoin, G. et al. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat Chem Biol 11, 728–732 (2015). https://doi.org/10.1038/nchembio.1879

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