Shimada Norimoto, Sasaki Ryohsuke, Sato Shusei, Kaneko Takakazu, Tabata Satoshi, Aoki Toshio, Ayabe Shin-ichi
Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan.
J Exp Bot. 2005 Sep;56(419):2573-85. doi: 10.1093/jxb/eri251. Epub 2005 Aug 8.
Dihydroflavonol 4-reductase (DFR) is the first committed enzyme of the anthocyanin and condensed tannin pathways. Several DFR cDNAs have been cloned, and different specificities of DFR isozymes in the substrate hydroxylation patterns have been reported, but only fragmentary knowledge of DFR gene organization is available. Reported here is a comprehensive analysis of DFRs of a model legume, Lotus japonicus. A total of five DFR genes were found to form a cluster within a 38 kb region in the L. japonicus genome, whereas six cDNAs, including two splicing variants resulting from a transversion at a splicing acceptor site, were cloned. All the genes were expressed, with different organ specificities, in the mature plant. Three of the DFR proteins heterologously expressed in Escherichia coli showed catalytic activity, and their substrate preferences agreed with the variation of a specific active site residue (Asp or Asn) reported to control the specificity. The hydroxylation patterns of anthocyanidins and condensed tannin units in the stems did not reflect the substrate specificity of the expressed isozymes, implying complex regulation mechanisms in the biosynthesis. The two splicing variants and one DFR with Ser at the specificity-controlling position failed to show the activity, but a revertant protein replacing the unusual splicing restored the activity. The phylogenetic tree, constructed with known DFR sequences, showed evolutionary divergence of some of the DFR genes prior to the plant speciation. This work affords the basis for genetic and biochemical studies on the diversity of DFR and the flavonoid products.
二氢黄酮醇4-还原酶（DFR）是花青素和缩合单宁途径中的第一个关键酶。已经克隆了几个DFR cDNA，并且报道了DFR同工酶在底物羟基化模式上的不同特异性，但关于DFR基因组织的知识仅为片段性的。本文报道了对模式豆科植物百脉根DFR的全面分析。在百脉根基因组中一个38 kb的区域内共发现五个DFR基因形成一个簇，同时克隆了六个cDNA，包括由于剪接受体位点的颠换产生的两个剪接变体。所有这些基因在成熟植株中均有表达，且具有不同的器官特异性。在大肠杆菌中异源表达的三种DFR蛋白显示出催化活性，它们对底物的偏好与据报道控制特异性的一个特定活性位点残基（天冬氨酸或天冬酰胺）的变异一致。茎中花青素和缩合单宁单元的羟基化模式并未反映所表达同工酶的底物特异性，这意味着生物合成中存在复杂的调控机制。两个剪接变体以及在特异性控制位置含有丝氨酸的一个DFR未能显示出活性，但一个取代异常剪接的回复蛋白恢复了活性。用已知的DFR序列构建的系统发育树表明，一些DFR基因在植物物种形成之前就发生了进化分歧。这项工作为DFR和类黄酮产物多样性的遗传和生化研究提供了基础。
