Identification and Expression of Genes Involved in the Conversion of Daidzein and Genistein by the Equol-Forming Bacterium Slackia isoflavoniconvertens
| Autor: | Anastasia Matthies, Wolfram Engst, Annett Braune, Michael Blaut, Christine Schröder |
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| Rok vydání: | 2013 |
| Předmět: |
Molecular Sequence Data
Sequence Homology Genistein Genetics and Molecular Biology Reductase Biology medicine.disease_cause Polymerase Chain Reaction Applied Microbiology and Biotechnology Chromatography Affinity chemistry.chemical_compound Escherichia coli medicine Humans Electrophoresis Gel Two-Dimensional Chromatography High Pressure Liquid chemistry.chemical_classification Base Sequence Ecology Daidzein food and beverages Sequence Analysis DNA Equol Isoflavones Molecular biology Actinobacteria Intestines Enzyme chemistry Biochemistry Heterologous expression Oxidoreductases Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 79:3494-3502 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.03693-12 |
| Popis: | Gut bacteria play a key role in the metabolism of dietary isoflavones, thereby influencing the availability and bioactivation of these polyphenols in the intestine. The human intestinal bacterium Slackia isoflavoniconvertens converts the main soybean isoflavones daidzein and genistein to equol and 5-hydroxy-equol, respectively. Cell extracts of S. isoflavoniconvertens catalyzed the conversion of daidzein via dihydrodaidzein to equol and that of genistein to dihydrogenistein. Growth of S. isoflavoniconvertens in the presence of daidzein led to the induction of several proteins as observed by two-dimensional difference gel electrophoresis. Based on determined peptide sequences, we identified a cluster of eight genes encoding the daidzein-induced proteins. Heterologous expression of three of these genes in Escherichia coli and enzyme activity tests with the resulting cell extracts identified the corresponding gene products as a daidzein reductase (DZNR), a dihydrodaidzein reductase (DHDR), and a tetrahydrodaidzein reductase (THDR). The recombinant DZNR also converted genistein to dihydrogenistein at higher rates than were observed for the conversion of daidzein to dihydrodaidzein. Higher rates were also observed with cell extracts of S. isoflavoniconvertens . The recombinant DHDR and THDR catalyzed the reduction of dihydrodaidzein to equol, while the corresponding conversion of dihydrogenistein to 5-hydroxy-equol was not observed. The DZNR, DHDR, and THDR were expressed as Strep -tag fusion proteins and subsequently purified by affinity chromatography. The purified enzymes were further characterized with regard to their activity, stereochemistry, quaternary structure, and content of flavin cofactors. |
| Databáze: | OpenAIRE |
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