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

植物积累各种天然产物。它们通过多步酶反应过程生物合成。已经设想了在植物细胞中有效合成特定产物的一些机制，例如酶复合物。异黄酮主要在豆科植物中生物合成，在生产植物中起重要作用。本研究利用功能表达分级筛选的方法，从刺根、百脉根和大豆中克隆了植物抗毒素合成途径中的2-羟基异黄烷酮4 '-O-甲基转移酶（HI 4'OMT）和2-羟基异黄烷酮脱氢酶（HID）的cDNA，以及植物抗毒素合成途径中紫檀烷还原酶（PTR）的cDNA，RT-PCR和EST信息：聚酮还原酶（PKR）参与6 ′-脱氧查耳酮的形成。我们证明了重组棘球蚴PKR蛋白还介导棘球蚴苷生物合成的后期步骤，即2 '-O-甲基吡啶二酮还原。设想棘球蛋白生物合成中PKR的酶循环。用OMT亲和层析柱对刺灵芝HI 4 'OMT进行了部分纯化，HID蛋白与HI 4'OMT共洗脱，表明HI 4 'OMT与HID蛋白之间存在相互作用。最后，为了阐明酶在体内的功能，进行了将IFS和HID cDNA整合到重组酵母系统中的实验。同时表达IFS和HID的酵母细胞比单一IFS底物积累了更高水平的来自外源性黄烷酮的异黄酮。据推测，IFS和HID之间的合作是需要有效的合成荧光素。

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.