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Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes

An Erratum to this article was published on 01 May 2000

Abstract

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

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Figure 1: A simplified diagram of the phenylpropanoid pathway showing intermediates and enzymes involved in isoflavone synthesis.
Figure 2: Genistein synthesis in assays of yeast microsomes containing IFS1.
Figure 3: Daidzein synthesis in assays of yeast microsomes containing IFS1.
Figure 4: Comparison of daidzein and genistein synthesis by IFS1 and IFS2.
Figure 5: Assays of Arabidopsis plant extracts.

Notes

  1. The online full-text version of this article reflects an editorial correction to this sentence that has not been corrected in the PDF and print versions.

References

  1. Nestel, P.J., et al. Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal women. J. Clin. Endocrinol. Metab. 84, 895–898 ( 1999).

    CAS  PubMed  Google Scholar 

  2. Murkies, A.L. et al. Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat. Maturitas 21, 189 –195 (1995).

    Article  CAS  Google Scholar 

  3. Civitelli, R. In vitro and in vivo effects of ipriflavone on bone formation and bone biomechanics. Calcif. Tissue Int. 61, Suppl: S12–14 (1997).

    Article  CAS  Google Scholar 

  4. Gennari, C. et al. Effects of ipriflavone—a synthetic derivative of natural isoflavones—on bone mass in early years after menopause. Menopause 5, 9–15 (1998).

    CAS  PubMed  Google Scholar 

  5. Sharma, R.D. Isoflavones and hypercholesterolemia in rats. Lipids 14, 535–540. (1979).

    Article  CAS  Google Scholar 

  6. Peterson, G. & Barnes, S. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem. Biophys. Res. Commun. 179, 661–667 (1991).

    Article  CAS  Google Scholar 

  7. Messina, M. & Barnes, S. The role of soy products in reducing cancer risk. J. Natl. Cancer Inst. 83, 541 –546 (1991).

    Article  CAS  Google Scholar 

  8. Food labeling: health claims; soy protein and coronary heart disease; final rule. Federal Register 64 FR 57699, October 26, 1999. (http://www.fda.gov/).

  9. Tsukamoto, C. et al. Factors affecting isoflavone content in soybean seeds: changes in isoflavones, saponins and composition of fatty acids at different temperatures during seed development. J. Agric. Food Chem. 43, 1184–1192 (1995).

    Article  CAS  Google Scholar 

  10. Eldridge, A.C. & Kwolek, W.F. Soybean isoflavones: effect of environment and variety on composition. J. Agric. Food Chem. 31, 394–396 ( 1983).

    Article  CAS  Google Scholar 

  11. Wang, H.-J & Murphy, P.A. Mass balance study of isoflavones during soybean processing. J. Agric. Food Chem. 44, 2377–2383 (1996).

    Article  CAS  Google Scholar 

  12. Okubo, K., et al. Components responsible for the undesirable taste of soybean seeds. Bioscience. Biotechnol. Biochem. 56, 99– 103 (1992).

    Article  CAS  Google Scholar 

  13. Padmavati, M. & Reddy, A.R. Flavonoid biosynthetic pathway and cereal defence response: an emerging trend in crop biotechnology. Plant Biochem. Biotechnol. 8, 15–20 (1999).

    Article  CAS  Google Scholar 

  14. Dixon, R.A. & Pavia, N.L. Stress-induced phenylpropanoid metabolism . Plant Cell 7, 1085–1097 (1995).

    Article  CAS  Google Scholar 

  15. Blount, J.W., Dixon, R.A. & Paiva, N.L. Stress response in alfalfa (Medicago sativa L.). XVI. Antifungal activity of medicarpin and its biosynthetic precursors: implications for the genetic manipulation of stress metabolites. Physiol. Mol. Plant Pathol. 41, 333– 349 (1992).

    Article  CAS  Google Scholar 

  16. Graham, T.L. in Handbook of phytoalexins metabolism and action (eds Daniel, M. & Purkayastha, R.P.) 85–116 (Marcel Dekker, New York; 1995).

  17. Ebel, J. Phytoalexin synthesis: the biochemical analysis of the induction process. Annu. Rev. Phytopathol. 24, 235–264 (1986).

    Article  CAS  Google Scholar 

  18. Rivera-Vargas, L.I., Schmitthenner, A.F. & Graham, T.L. Soybean flavonoid effects on and metabolism by Phytophthora sojae. Phytochemistry 32, 851–857 (1993).

    Article  CAS  Google Scholar 

  19. Graham, T.L. & Graham, M.Y. in Plant–microbe interactions . (eds Keen, N. & Stacey, G.) (APS Press, St. Paul; 2000), in press.

  20. Pueppke, J.L. The genetics and biochemical basis for nodulation of legumes by rhizobia. Crit. Rev. Biotechnol. 16, 1–51 (1996).

    Article  CAS  Google Scholar 

  21. Hashim, M.F., Hatkamatsuka, T., Ebizuka, Y. & Sankawa, U. Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis . FEBS Lett. 271, 219–222 (1990).

    Article  CAS  Google Scholar 

  22. Kochs, G. & Griesbach, H. Enzymic synthesis of isoflavones . Eur. J. Biochem. 155, 311– 318 (1986).

    Article  CAS  Google Scholar 

  23. Schopfer, C.R. & Ebel, J. Identification of elicitor-induced cytochrome p450s of soybean (Glycine max L.) using differential display of mRNA. Mol. Gen. Genet. 258, 315–322 (1998).

    Article  CAS  Google Scholar 

  24. Bolwell, G.P., Bozac, K. & Zimmerlin, A. Plant cytochrome P450. Phytochemistry 37, 1491–1506 (1994).

    Article  CAS  Google Scholar 

  25. Pompon, D., Louerat, B., Bronne, A. & Urban, P. Yeast expression of animal and plant p450s in optimized redox environments. Methods Enzymol. 272, 51–64 ( 1996).

    Article  CAS  Google Scholar 

  26. Nature Biotechnology Web Extras site ( http://biotech.nature.com/web_extras/).

  27. Siminszky, B., Corbin, F.T., Ward, E.R., Fleischmann, T.J. & Dewey, R.E. Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides . Proc. Natl. Acad. Sci. USA 96, 1750– 1755 (1999).

    Article  CAS  Google Scholar 

  28. Steele, C.L., Gijzen, M., Qutob, D. & Dixon, R.A. Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch. Biochem. Biophys. 367, 146–150 (1999).

    Article  CAS  Google Scholar 

  29. Dewick, P.M. in The flavonoids: advances in research. (eds Harborne, J.B. & Mabry, T.J.) 535–640 (Chapman and Hall, New York; 1982).

  30. Geigert, J., Stermitz, F.R., Johnson, G., Maag, D.D. & Johnson, D.K. Two phytoalexins from sugarbeet (Beta vulgaris) leaves. Tetrahedron 29, 2703–2706 (1973).

    Article  CAS  Google Scholar 

  31. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 ( 1990).

    Article  CAS  Google Scholar 

  32. Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1, 19–27 (1989).

    Article  Google Scholar 

  33. Johnston, M. & Davis, R.W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1440–1448 ( 1984).

    Article  CAS  Google Scholar 

  34. Hua, S.B., Qiu, M., Chan, E., Zhu, L. & Luo, Y. Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 38, 91– 96 (1997).

    Article  CAS  Google Scholar 

  35. Hasenfratz, M.P. Clonage de la NADPH-cytochrome P450 reductase et d'une proteine calnexine-like chez Helianthus tuberosus. (Universite Louis Pasteur, Strasbourg, France; 1992).

  36. Odell, J.T., Nagy, F. & Chua, N.-H Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313, 810–812 (1985).

    Article  CAS  Google Scholar 

  37. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. & Goodman, H.M. Nopaline synthase: transcript mapping and DNA sequence . J. Mol. Appl. Genet. 1, 561– 573 (1982).

    CAS  PubMed  Google Scholar 

  38. Hajdukiewicz, P., Svab, Z. & Maliga, P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994 (1994).

    Article  CAS  Google Scholar 

  39. Bechtold, N., Ellis, J. & Pelletier, G. In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sci. 316, 1194– 1199 (1993).

    CAS  Google Scholar 

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Acknowledgements

Thanks to Alfred A. Ciuffetelli, Tina Henry-Smith and June Shi for technical help; Edgar Cahoon, Sean J. Coughlan, and David Styles for analytical help; and Mike Hanafey and Mike Ramaker for bioinformatics support. Scott Tingey, Guo-Hua Miao, and Maureen Dolan are responsible for the DuPont Genomics Program, which provided invaluable source material. Thanks also to Wolfgang Shuh (Pioneer Hibred International) for the fungally treated soybean tissue, Pal Maliga (Waksman Institute, Rutgers University) for the binary vector and Daniele Werck-Reichhart (CNRS- Strasbourg) for construction of WHT1. Thanks to Enno Krebbers and Bill Hitz for thoughtful discussion.

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Correspondence to Brian McGonigle.

Supplementary information

Relationship of IFS1 to other cytochromes P450

The cDNA identified by this functional screening as encoding a protein with isoflavone synthase activity was fully sequenced and shown to be closely related to cyp93c1, a soybean cDNA with no reported enzyme activity (accession number AF022462; ref. 1). Cytochromes P450 are named on the basis of structural similarity, not functional similarity (http://drnelson.utmem.edu/CytochromeP450.html), although proteins with a high degree of structural similarity may be expected to have some functional relatedness. Cytochrome P450 proteins that are greater then 40% identical are placed in the same family, while proteins that are greater then 55% identical are placed in the same subfamily. The cyp93 family has thirteen members, divided into four subfamilies, for which full-length sequences are deposited with the cytochrome P450 nomenclature committee (http://drnelson.utmem.edu/CytochromeP450.html). The cyp93a subfamily has four members, only one of which has been functionally identified. One of these genes, isolated from soybean, encodes dihydroxypterocarpan 6a-hydroxylase, an enzyme in the pathway that converts daidzein to glyceollins2. The cyp93b subfamily has five members, one of which has been functionally identified. Cyp93b1, isolated from licorice, encodes (2S)-flavanone 2-hydroxylase. This enzyme converts the hydroxyflavanones naringenin and liquiritigenin, the same substrates used by IFS, to flavones3. The cyp93c subfamily has three members other than cyp93c1, none of which has been functionally identified. The cyp93d subfamily has only one member, which has been identified through genomic sequencing of Arabidopsis. Neither a cDNA nor an enzymatic activity has been reported for this genomic sequence. The identification of IFS as being a member of the cyp93 family is consistent with the roles that the other characterized members of this family play in the phenylpropanoid pathway. Though IFS1 and cyp93b1 act on the same substrates, they have only 48% amino acid identity.

References to Supplementary information

  1. B1

    Siminszky, B., Corbin, F.T., Ward, E.R., Fleischmann, T.J. & Dewey, R.E. Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. . Proc. Natl. Acad. Sci. U.S.A., 96 1750-1755 (1999).

  2. B2

    Schopfer, C.R., Koch, G., Lottspeich, F. & Ebel, J. Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.). FEBS Lett., 432, 182-186 (1998).

  3. B3

    Akashi, T., Aoki, T. & Ayabe, S. Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II. . FEBS Lett., 431, 287-290 (1998).

Supplementary figures

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Figure 12 (GIF 65.6 KB)

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Jung, W., Yu, O., Lau, SM. et al. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nat Biotechnol 18, 208–212 (2000). https://doi.org/10.1038/72671

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