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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的氧化还原过程。电子载体蛋白、铁还蛋白(Fd)和依赖于Fd的酶结合在一起，就像是这种代谢的分子机制。电子通过FD分配给叶绿体内各种酶的过程是光合作用活性的关键，阐明其分子机制是本研究的主要目的。研究结果如下：1)测定了根ED和FD的电子转移络合物的X射线晶体结构，发现该结构不同于LEF的结构。2)用野生型ANS研究了亚硫酸盐还原酶和FD之间的电子转移，发现FD的突变分子和氧化还原电位对亚硫酸盐还原酶的活性和底物专一性有重要影响。对该酶进行了X射线晶体分析。3)对依赖FD的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。它们在电子转移到光系统中的能力。I、FD：NADP+还原酶和亚硫酸盐还原酶有显著差异，并阐明了它们的生理分化作用。5)缺乏依赖FD的谷氨酸合成酶的蓝藻突变体表现出碳氮同化失衡，这可能是由于还原当量的分配受到干扰所致。为了应对这种失衡，谷氨酸合成酶基因的表达受到了上调和下调。我们可以通过报告分析系统来监测该基因的表达。