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Study on the molecular basis for the regulation of redox metabolism network in plastids

质体氧化还原代谢网络调控的分子基础研究

基本信息

批准号：
13440240
负责人：
HASE Toshiharu
金额：
$ 10.75万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Autotrophic capacity of plants depends on a series of redox of redox metabolisms. An electron carrir protein, ferredoxin (Fd) and Fd-dependent enzymes function in combination as a molecular machinery for such metabolisms. The process of electron partitioning via Fd to various enzymes within plastids is crucial for photosynthetic activity and elucidation of its molecular mechanism is the main aim of this study. Following results has been obtained.1) X-ray crystsl structure of an electron transfer complex of root Ed and Fd:NADP+ reductase has been determied and this structure is found to be different from that of lesf counterpart.2) Electron transfer between sulfite reductase and Fd was investigate using wild type ans their mutant molecules and redox potential of Fd was found to be important for activity and substrate specificity of sulfite reductase. X-ray crystal analysis of this enzyme is now ongoing.3) Site diredted mutagenesis study of Fd-dependent glutamate synthase was done. Amino acids necessary for the activity of he amidotransferae domain of this enzyme were identified.4) Based on the total total genome information of Arabidopsis, 4 Fd isoprotiens were prepared and purified by reconbinant technique. Their abilities in the electron transfer to photosystem. I, Fd:NADP+ reductase and sulfite reductase were significantly different and physiological differentiation of their role was elucidated. Now proteins which interact with each Fd isoprotein are under investigation.5) A cyanobacterial mutant lacking Fd-dependent glutamate synthase exhibits unbalancing of carbon and nitrogen assimilation probably due to disturbance of partitioning of reducing equivalent. In response to this unbalancing the expression of glutamate synthase gene was up and down-regulated. We could monitor this gene expression by reporterassay syatem.

植物的自养能力取决于一系列氧化还原代谢过程。电子载体蛋白、铁氧还蛋白（Fd）和Fd依赖性酶作为这种代谢的分子机制组合起作用。质体内电子通过Fd分配给各种酶的过程是光合活性的关键，阐明其分子机制是本研究的主要目的。获得了以下结果。1）测定了根艾德和Fd的电子转移复合物的X射线晶体结构：NADP+还原酶，发现该结构与相应物的结构不同。2）使用野生型及其突变体分子研究了亚硫酸盐还原酶和Fd之间的电子转移，发现Fd的氧化还原电位对亚硫酸盐还原酶的活性和底物特异性很重要。艾德：NADP+还原酶。3）对Fd依赖型谷氨酸合成酶进行了定点突变研究。4）根据拟南芥全基因组信息，利用重组技术制备并纯化了4个Fd同源蛋白。它们将电子传递到光系统的能力。I、Fd：NADP+还原酶和亚硫酸盐还原酶存在显著差异，阐明了它们作用的生理分化。目前正在研究与Fd同工酶相互作用的蛋白质。5）一个缺乏Fd依赖的谷氨酸合酶的蓝藻突变体表现出碳氮同化的不平衡，可能是由于还原当量分配的干扰。谷氨酸合成酶基因的表达随着这种不平衡而上调和下调。我们可以通过荧光定量分析系统来监测该基因的表达。