- Article
- Published:
- Woosuk Jung1,
- Oliver Yu1,
- Sze-Mei Cindy Lau2,
- Daniel P. O'Keefe2,
- Joan Odell1,
- Gary Fader1 &
- …
- Brian McGonigle1
Nature Biotechnology volume18,pages 208–212 (2000)Cite this article
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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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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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Authors and Affiliations
Agricultural Biotechnology, The Dupont Company, Experimental Station, PO Box 80402, Wilmington, DE 19880-0402
Woosuk Jung,Oliver Yu,Joan Odell,Gary Fader&Brian McGonigle
Central Research and Development, The DuPont Company, Experimental Station, P.O. Box 80402, Wilmington, DE 19880-0402
Sze-Mei Cindy Lau&Daniel P. O'Keefe
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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
- 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).
- 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).
- 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).
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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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DOI: https://doi.org/10.1038/72671
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