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

植物积累了多种天然产物。它们是通过多步酶反应过程生物合成的。已经设想了一些机制，例如酶复合物，在植物细胞中有效地合成特定的产物。异黄酮类化合物主要在豆科植物中进行生物合成，在生产植物中起着重要作用。在本研究中，我们克隆了该途径的编码酶的cdna，并试图证明其生物合成的机制。利用功能表达分离筛选、RT-PCR和EST信息，从草甘膦、荷花和大豆中克隆了参与异黄酮生物合成的2-羟基异黄酮4′- o -甲基转移酶（hi4′-O-methyltransferase, hi4′-O-methyltransferase, hi4′-O-methyltransferase, hi4′-O-methyltransferase, hi4′- omt）和2-羟基异黄酮脱水酶（hi4′- omt）和植物抗逆素途径的pterocarpan reductase (PTR) cdna。聚酮还原酶（PKR）参与6'-脱氧查尔酮的形成。我们发现重组棘球菊PKR蛋白也介导了棘球菊素生物合成的后期步骤，即2′- o -甲基氯二酮还原。展望了PKR在紫锥素生物合成中的酶循环利用。采用OMT特异性亲和柱对棘球菊hi4’OMT进行部分纯化。HID蛋白与HI4'OMT共洗脱，提示HI4'OMT与HID存在蛋白蛋白相互作用。最后，为了阐明该酶在体内的功能，我们在重组酵母系统中加入IFS和HID cdna进行了实验。同时表达IFS和HID的酵母细胞从外源黄酮中积累的异黄酮类化合物水平高于单一IFS转化的酵母细胞。推测IFS和HID的协同作用是有效合成异黄酮的必要条件。

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.