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

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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.