Molecular Evolution of Denitrifying Systems of Fungi and Actinomycetes

(1)尖孢镰刀菌（Fusarium oxysporum）和tonkinense圆柱碳菌（cydrocarpon tonkinense）的反硝化系统定位于线粒体，在与ATP合成偶联的反硝化线粒体中检测到硝酸盐还原酶和亚硝酸盐还原酶等反硝化酶活性。结果导致在真菌线粒体中发现厌氧呼吸系统。我们还通过电镜和免疫荧光染色观察了反硝化条件下真菌细胞的线粒体，发现即使在这种缺氧条件下，真菌线粒体也正常进展，反硝化系统定位于线粒体。(2)细胞色素P45Onor虽然属于P450超家族，但它催化的反应和P450一样独特。我们成功地测定了P450nor的晶体结构，为阐明其反应机理提供了很大的进展。(3)虽然我们在真菌中发现了更多的denitr活性，但在酵母和放线菌等重要微生物类群中没有发现这种活性。因此，我们对这些微生物的活性进行了筛选，在酵母和放线菌中发现了许多反硝化菌。我们明确了放线菌硫链霉菌、酵母皮毛霉和福州霉的反硝化系统，发现了T.cutaneum中存在P450nor，并从S.thioluteus中纯化了亚硝酸盐还原酶（Nir）和azurin。(4)分离并鉴定了真菌反硝化系统中尚未分离到的硝酸盐还原酶（Nar）、黄血红蛋白和细胞色素c_<549>，以及c . tonkinensis的Nir等成分，首次证实了真菌、酵母和放线菌中存在反硝化活性。真核生物的反硝化系统被证明定位于线粒体与ATP合成的偶联，从而起到厌氧呼吸作用。这些结果不仅表明线粒体中存在一种新的呼吸系统，而且还说明了呼吸系统进化的重要方面和问题

(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