Molecular Evolution of Denitrifying Systems of Fungi and Actinomycetes

(1)尖孢镰刀菌（Fusarium oxysporum）和东京赤霉病菌（Aciddrocarpon tonkinense）的厌氧呼吸系统均位于线粒体内，并在线粒体内检测到与ATP合成偶联的硝酸还原酶和亚硝酸还原酶活性，从而证实了尖孢镰刀菌（Fusarium oxysporum）和东京赤霉病菌（Aciddrocarpon tonkinense）的厌氧呼吸系统。我们还通过电子显微镜以及免疫荧光染色观察了在脱水条件下生长的真菌细胞中的线粒体，并且发现即使在这种氧限制条件下真菌线粒体也正常进行，并且脱水系统定位于线粒体。(2)细胞色素P45Onor虽然属于P450超家族，但催化的反应与P450一样独特。我们成功地确定了P450nor的晶体结构，这为阐明其反应机理提供了一个很大的进展。(3)尽管我们发现了 关于我们 在真菌中发现了这种活性，但在重要的微生物类群、酵母和放线菌中没有发现这种活性。因此，我们对这些微生物进行了活性筛选，在酵母菌和放线菌中发现了许多细菌。本研究阐明了放线菌Streptomyces thioluteus、酵母Trichosporon cutaneum和Fellomyces fuzhouensis的发酵体系，发现皮丝孢酵母中存在P450nor，并从硫链霉菌中纯化了亚硝酸盐还原酶（Nir）和天青蛋白。(4)分离并鉴定了迄今为止尚未分离到的真菌发酵系统的组分，如尖孢镰刀菌的硝酸还原酶（Nar）、黄素血红蛋白、细胞色素<549>c_2和越南刺盘孢菌的Nir，首次证明了真菌、酵母和放线菌之间均存在发酵活性。真核生物的呼吸系统被证明定位于与ATP合成偶联的线粒体上，从而起到无氧呼吸的作用。这些结果不仅提出了一个新的呼吸系统的存在，在南极洲，但也重要的方面和主题要阐明有关的呼吸系统的演变减

(1) The denitrifying systems of the fungus Fusarium oxysporum and Cylindrocarpon tonkinense were shown to be localized at the mitochondrion and denitrifying enzyme activities such as nitrate reductase and nitrite reductase activities were detected in the denitrifying mitochondria coupling to the synthesis of ATP.The results resulted in the finding of an anaerobic respiration system in the fungal mitochondrion. We observed the mitochondria in the fungal cells grown under denitrifying conditions also by electron microscopy as well as the immuno-fluorescent staining, and found that the fungal mitochondria are normally progressed even under such oxygen-restricted conditions and denitrifying systems are localized to the mitochondria.(2) Cytochrome P45Onor catalyzes a unique reaction as P450 although it belongs to the P450 superfamily. We succeeded in determining the crystal structure of P450nor, which afforded a great advance in clarifying its reaction mechanism.(3) Although we found denitr … More ifying activities in fungi, such activities had not been found in important microbial taxa, yeasts and actinomycetes. So we screened the activities among those microorganisms and found many denitrifiers in yeasts and actinomycetes. We clarified the denitrifying systems of the actinomycete Streptomyces thioluteus and the yeasts Trichosporon cutaneum and Fellomyces fuzhouensis, We found the occurrence of P450nor in T.cutaneum and purified nitrite reductase (Nir) and azurin from S.thioluteus.(4) We isolated and charactgerized components of the denitrifying systems of fungi that were not isolated so far, such as nitrate reductase (Nar), flavohemoglobin, and cytochrome c_<549> from F.oxysporum and Nir from C.tonkinense.These results demonstrated for the first time the occurrence of denitrifying activities among fungi, yeasts, and actinomycetes. The eukaryotic denitrifying systems were shown to be localized at the mitochondrion coupling to the synthesis of ATP and thus act as an anaerobic respiration. These results presented not only the existence of a novel respiration system in the mitochondrion but also important aspects and subjects to be elucidated concerning the evolution of the respiratory systems Less