Molecular biological analysis of the enzyme networks using the legume isoflavonoid pathway as a model system

使用豆类异黄酮途径作为模型系统对酶网络进行分子生物学分析

基本信息

批准号：
15510183
负责人：
AKASHI Tomoyoshi
金额：
$ 2.11万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Plants accumulate a variety of natural products. They are biosynthesized by multi-step enzyme reaction processes. Some mechanisms, e.g. enzyme complex, for effective synthesis of specific products in plant cells have been envisaged. Isoflavonoids are biosynthesized mainly in leguminous plants and play important roles in the producer plants. In this study, we cloned cDNAs encoding enzymes of the pathway and attempted to demonstrate the mechanism for the biosynthesis.cDNAs of 2-hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) and 2-hydroxyisoflavanone dehydratase (HID) involved in isoflavone biosynthesis and cDNAs of pterocarpan reductase (PTR) in phytoalexin pathway were cloned from Glycyrrhiza echinata, Lotus japonicus and soybean using functional expression fractionation screenings, RT-PCR and EST information.Polyketide reductase (PKR) is involved in 6'-deoxychalcone formation. We demonstrated that the recombinant G.echinata PKR protein also mediates the late step of echinatin biosynthesis, i.e.2'-O-methyllicodione reduction. Enzyme recycling of PKR in echinatin biosynthesis is envisaged. Partial purification of G.echinata HI4'OMT was performed using an OMT specific affinity column.HID protein was co-eluted with HI4'OMT, suggesting the protein-protein interaction between HI4'OMT and HID. Finally, to clarify the enzyme function in vivo, experiments incorporating IFS and HID cDNAs in recombinant yeast systems were carried out. The yeast cells expressing both IFS and HID accumulated higher levels of isoflavonoids from exogenously supplied flavanones than the single IFS transformant. It is assumed that cooperation between IFS and HID is required for effective synthesis of isoflavonoids.

植物积累了各种天然产品。它们是通过多步酶反应过程生物合成的。一些机制，例如已经设想了为有效合成植物细胞中特定产物的酶复合物。异黄素主要在豆科植物中生物合成，并在生产植物中起重要作用。在这项研究中，我们克隆了编码途径酶的cDNA，并试图证明生物合成的机制。2-羟基异氟酮4'-o-甲基转移酶（HI4'OMT）的CDNA，与2-羟基异氟烷酮的cdnose and cdn osfnawase（HI4'OMT）涉及Isoftasase（Hiddnose）（HID）。使用功能表达分级筛选，RT-Polyketide sefigners，plosekydide Reeductase（PKR），参与6'-二氧基chalcone的形式。我们证明了重组G.echinata PKR蛋白还介导了echinatin生物合成的后期步骤，即还原。设想了echinatin生物合成中PKR的酶回收。使用OMT特异性亲和力柱进行G.echinata Hi4'omt的部分纯化。HID蛋白与HI4'OMT共洗脱，表明HI4'OMT和HID之间的蛋白质蛋白质相互作用。最后，为了阐明体内的酶功能，进行了重组酵母系统中的IF和HID cDNA的实验。表达IFS和HID的酵母菌细胞比单个IFS转化的外源提供的黄酮中积累了更高水平的异黄酮。假定有效合成异黄酮类似IFS和HID之间的合作是必需的。