Daidzein reductase of Eggerthella sp. YY7918, its octameric subunit structure containing FMN/FAD/4Fe-4S, and its enantioselective production of R-dihydroisoflavones
| Autor: | Emiko Yanase, Yuika Kawada, Rie Sawamura, Keiichi Yamaguchi, Mizuho Inagaki, Tomoko Goshima, Shin-ichiro Yokoyama, Toshio Niwa, Osamu Sakurada, Yuta Kato, Kazuo Kuwata, Tohru Suzuki, Akio Ebihara |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Oxidoreductases Acting on CH-CH Group Donors Flavin Mononucleotide Iron 030106 microbiology Coenzymes Genistein Bioengineering Reductase Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound Escherichia coli Humans Formononetin Enzyme kinetics Protein Structure Quaternary biology Chemistry Daidzein food and beverages Stereoisomerism Glycitein Equol Isoflavones Enzyme assay Actinobacteria Protein Subunits Biochemistry Flavin-Adenine Dinucleotide biology.protein Protein Multimerization Oxidoreductases NADP Sulfur Biotechnology |
| Zdroj: | Journal of Bioscience and Bioengineering. 126:301-309 |
| ISSN: | 1389-1723 |
| DOI: | 10.1016/j.jbiosc.2018.03.018 |
| Popis: | S-Equol is a metabolite of daidzein, a type of soy isoflavone, and three reductases are involved in the conversion of daidzein by specific intestinal bacteria. S-Equol is thought to prevent hormone-dependent diseases. We previously identified the equol producing gene cluster (eqlABC) of Eggerthella sp. YY7918. Daidzein reductase (DZNR), encoded by eqlA, catalyzes the reduction of daidzein to dihydrodaidzein (the first step of equol synthesis), which was confirmed using a recombinant enzyme produced in Escherichia coli. Here, we purified recombinant DZNR to homogeneity and analyzed its enzymological properties. DZNR contained FMN, FAD, and one 4Fe-4S cluster per 70-kDa subunit as enzymatic cofactors. DZNR reduced the C C bond between the C-2 and C-3 positions of daidzein, genistein, glycitein, and formononetin in the presence of NADPH. R-Dihydrodaidzein and R-dihydrogenistein were highly stereo-selectively produced from daidzein and genistein. The Km and kcat for daidzein were 11.9 μM and 6.7 s−1, and these values for genistein were 74.1 μM and 28.3 s−1, respectively. This enzyme showed similar kinetic parameters and wide substrate specificity for isoflavone molecules. Thus, this enzyme appears to be an isoflavone reductase. Gel filtration chromatography and chemical cross-linking analysis of the active form of DZNR suggested that the enzyme consists of an octameric subunit structure. We confirmed this by small-angle X-ray scattering and transmission electron microscopy at a magnification of ×200,000. DZNR formed a globular four-petal cloverleaf structure with a central vertical hole. The maximum particle size was 173 A. |
| Databáze: | OpenAIRE |
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