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

植物积累了各种各样的天然产物。它们是通过多步酶反应过程生物合成的。已经设想了一些在植物细胞中有效合成特定产物的机制，例如酶复合体。异黄酮类化合物主要在豆科植物中合成，在生产型植物中发挥着重要作用。本研究利用功能表达片段筛选、RT-PCR和EST信息，克隆了异黄酮类生物合成途径中的2-羟基异黄酮4‘-O-甲基转移酶(HI4’OMT)和2-羟基异黄酮脱水酶(HID)的cDNA，并克隆了植物防御素途径中的蝶形烃还原酶(PTR)的cDNA。我们证明了重组棘球藻蛋白受体蛋白也参与了棘球蛋白生物合成的后期步骤，即2‘-O-甲基地衣二酮的还原。在儿茶素生物合成中，PKR的酶循环是被设想的。用OMT特异性亲和层析柱对棘球藻HI4‘OMT进行部分纯化，HID蛋白与HI4’OMT共洗脱，表明HI4‘OMT与HID之间存在蛋白质-蛋白质相互作用。最后，为了阐明该酶在体内的功能，在重组酵母系统中进行了掺入iFS和hID cDNA的实验。同时表达iFS和hid的酵母细胞从外源提供的黄烷酮中积累的异黄酮量高于单一的iFS转化子。假设异黄酮类化合物的有效合成需要IF和HID之间的合作。

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.