Biochemical and molecular biological studies of electron partitioning among enzymes involved in the assimilation of inorganic compounds in plants

参与植物无机化合物同化的酶之间电子分配的生化和分子生物学研究

基本信息

批准号：
05454019
负责人：
HASE Toshiharu
金额：
$ 4.48万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

1) Using heterologous expression system in E.coli, maize ferredoxin (Fd) was site-directly mutagenized. The substituted residues were designed surrounding [2Fe-2S] cluster and on the surface of molecule. The redox potential and electron transfer abilities to Fd-dependent enzymes, such as Fd-NADP reductase and sulfite reductase, were investigated and the result showed that Fd had a specific region to interact with the each enzyme.2) Two types of Fd isoproteins, photosynthetic and non-photosynthetic one, were expressed in a cyanobacterium, Plectonema boryanum, whose Fd gene (pet F) had been deleted. The transformant with photosynthetic Fd grew in photoautotrophic condition under light as the wild-type cells, while the transformant was not able to grow heterotrophically under dark. The transformant with non-photosynthetic Fd could not be selected probably due to the inability of this type of Fd to complement the Pet F.3) Genes for two glutamate synthase (GOGAT), Fd-GOGAT and NADH-GOGAT,were cloned from P.boryanum. The two GOGAT genes were separately inactivated by insertion of kanamycin cartridge. The resulting mutants were found to lack the corresponding enzymatic activity. However, the remarkable change f phenotype was not observed except for an increased sensitivity to photoinhibition. The two GOGATs are probably functionally compatible.

1）使用异源表达系统在大肠杆菌中，将玉米铁氧还蛋白（FD）直接定向诱变。取代的残基是设计围绕[2FE-2S]簇和分子表面的。 The redox potential and electron transfer abilities to Fd-dependent enzymes, such as Fd-NADP reductase and sulfite reductase, were investigated and the result showed that Fd had a specific region to interact with the each enzyme.2) Two types of Fd isoproteins, photosynthetic and non-photosynthetic one, were expressed in a cyanobacterium, Plectonema boryanum, whose Fd gene （PET F）已被删除。带有光合FD的转化剂在光线下像野生型细胞一样在光自营养状态下生长，而转化剂在黑暗下无法在异养中生长。无法选择具有非光合合成FD的转化剂，这可能是由于这种类型的FD无法补充两个谷氨酸合酶（GOGAT），FD-GOGAT和NADH-GOGAT的PET F.3）基因，它是从P.Boryanum中克隆的。通过插入卡纳米霉素墨盒分别灭活了两个GOGAT基因。发现所得的突变体缺乏相应的酶活性。但是，除了对光抑制的敏感性增加外，没有观察到显着的变化F表型。这两个Gogat可能在功能上兼容。