Identification and Expression of Genes Involved in the Conversion of Daidzein and Genistein by the Equol-Forming Bacterium Slackia isoflavoniconvertens
Identification and Expression of Genes Involved in the Conversion of Daidzein and Genistein by the Equol-Forming Bacterium Slackia isoflavoniconvertens
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DOI：
10.1128/aem.03693-12
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发表时间：
2013-06-01
影响因子：
4.4
通讯作者：
Braune, Annett
中科院分区：
文献类型：
--
作者：
Schroeder, Christine;Matthies, Anastasia;Braune, Annett
肠道细菌在膳食异黄酮的代谢中发挥关键作用，从而影响肠道中这些多酚的可利用性和生物活性。人肠道细菌Slackia isoflavonoids iconvertens可将大豆主要异黄酮大豆黄素和染料木素分别转化为雌马酚和5-羟基雌马酚。异黄酮细胞提取物催化大豆黄酮由二氢大豆黄酮转化为雌马酚，染料木素由二氢黄酮转化为染料木素。双向凝胶电泳结果显示，大豆苷元存在下，大豆异黄酮的生长诱导了几种蛋白质的产生。根据确定的肽序列，我们确定了一个由8个基因编码的大豆苷元诱导蛋白簇。将其中3个基因在大肠杆菌中进行异源表达，并用所得细胞提取物进行酶活性测试，鉴定出相应的基因产物为大豆苷元还原酶（DZNR）、二氢大豆苷元还原酶（DHDR）和四氢大豆苷元还原酶（THDR）。重组DZNR将染料木素转化为二氢异黄酮的速率也高于大豆苷元转化为二氢大豆苷元的速率。异黄酮类化合物的细胞提取物也观察到较高的率。重组DHDR和THDR催化双氢大豆苷元还原为雌马酚，而未观察到二氢异黄酮转化为5-羟基雌马酚。DZNR、DHDR和THDR以Strep-tag融合蛋白表达，通过亲和层析纯化。对纯化酶的活性、立体化学、四级结构和黄素辅助因子含量进行了进一步的表征。
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.