Molecular Evolution of Denitrifying Systems of Fungi and Actinomycetes

(1)尖孢镰刀菌和圆果孢子菌的反硝化系统定位于线粒体，反硝化酶活性如硝酸还原酶和亚硝酸还原酶活性与ATP的合成有关，因此发现真菌线粒体中存在厌氧呼吸系统。我们还通过电子显微镜和免疫荧光染色观察了在反硝化条件下生长的真菌细胞中的线粒体，发现真菌线粒体即使在这种限氧条件下也能正常进行，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，但它催化着与P450一样的独特反应。我们成功地测定了P450nor的晶体结构，这为阐明其反应机理提供了很大的进展。(3)虽然我们发现了脱硝基…在真菌中有更多的活性，在重要的微生物分类群、酵母和放线菌中没有发现这种活性。因此，我们对这些微生物的活性进行了筛选，在酵母和放线菌中发现了大量的反硝化细菌。我们对硫代链霉菌、皮肤毛孢霉和福州费氏酵母的反硝化系统进行了研究，在皮肤放线菌中发现了P450nor的存在，并从硫代链霉菌中分离纯化了亚硝酸还原酶(NIR)和天青素。(4)从真菌、酵母菌和放线菌中分离并鉴定了目前尚未分离到的真菌反硝化系统的组分，如硝酸还原酶(NAR)、黄色素、细胞色素C&lt；549&GT；真核生物的反硝化系统定位在线粒体上，与ATP的合成相耦合，从而起到厌氧呼吸的作用。这些结果不仅提出了线粒体中存在一种新的呼吸系统，而且还提出了关于呼吸系统进化的一些重要方面和有待阐明的主题。

(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