Molecular mechanism of multiple dissimilatory nitrogen metabolisms by eukaryotic and prokaryotic cells

(1)真菌反硝化系统的分子机制：从尖孢镰刀菌等真菌中分离到编码含铜亚硝酸还原酶(NirK)的基因，首次发现了真菌的反硝化活性。先前从尖孢镰刀菌中分离到的NirK蛋白被证明是NirK基因的产物。这是第一次从真核生物中分离出参与反硝化作用的细菌的同源物。系统发育分析表明，最近在许多真核微生物中发现的这些nirK基因及其同源基因都起源于单一祖先，表明它们来自原线粒体。研究表明，尖孢镰刀菌的硝酸盐还原系统由硝酸还原酶(DNAR，可能是细菌硝酸盐呼吸中普遍存在的NarGHI)和依赖泛醌的甲酸脱氢酶组成。与细菌系统不同，真菌DNAR系统被证明具有S…的功能在微好氧条件下更多地与细胞色素氧化酶结合，我们称之为混合呼吸。这些结果揭示了自然界中真菌反硝化作用的普遍存在。(2)细胞色素P450nor的反应机制：P450nor以一氧化氮(NO)还原酶的形式参与真菌反硝化作用，执行一种异常的电子转移，将2个电子从NADH直接转移到血红素蛋白。结构和动力学分析，如确定P450nor与NAD类似物的络合物的晶体结构，揭示了P450nor的详细反应机理，包括前手性氢化物从NADH转移到亚铁血红素-NO络合物的机理，以及关键光谱中间体的化学实体的鉴定。本研究结果将对生物无机化学和P450生物化学等诸多领域产生显著的影响。(3)真菌的氨发酵和厌氧生长：通过对真菌nidulans的uiaD或niia缺陷突变株的鉴定，证实了同化硝酸盐还原系统参与了真菌的氨发酵。研究表明，反硝化真菌尖孢镰刀菌能进行异乳酸发酵，并与氨发酵耦合。与反硝化作用的结果相结合的结果表明，至少有一部分土壤真菌是兼性的，而不是先前认为的专性好氧微生物。(4)其他主题：辅硝化作用是一种生物反应，而不是化学反应，这一结果得到了其对辅硝化底物的饱和动力学结果的支持，以及呼吸抑制剂(如氰化物)的抑制。放线菌被认为是专性需氧菌，相反，我们提供了放线菌抗生素链霉菌和天蓝色链霉菌存在硝酸盐呼吸的证据。为了将好氧反硝化菌应用于温室效应气体排放的控制，对其进行了分离，并对其性能进行了表征。较少

(1) Molecular mechanisms of fungal denitrification system: The genes encoding copper-containing nitrite reductase (nirK) were isolated from several fungi including Fusarium oxysporum, in which fungal denitrifying activity was first found. The NirK protein previously isolated from F. oxysporum, was shown to be the product of the nirK gene. This is the first isolation from eukaryote of an orthologoue of the bacterial counterpart involved in denitrification. Phylogenetic analysis shows that these nirK genes and their homologue genes that have been recently found in many eukaryotic microorganisms are originated from the single ancestor, suggesting that they are derived from the protomitochondrion. It was shown that the nitrate reducing system of F. oxysporum comprises nitrate reductase (dNaR, possibly NarGHI, which is universally found in bacterial nitrate respiration) and ubiquinone-dependent formate dehydrogenase. Unlike the bacterial system the fungal dNaR system was shown to function s … More imultaneously with cytochrome oxidase under micro-aerobic conditions, which we termed hybrid respiration. These results have revealed general occurrence of fungal denitrification in nature.(2) Reaction mechanism of cytochrome P450nor: P450nor participates in fungal denitrification as nitric oxide (NO) reductase, performing an unusual electron transfer, direct transfer of 2 electrons from NADH to the hemeprotein. Structural and kinetic analyses such as determination of the crystal structure of P450nor in complex with an NAD analogue revealed-the detailed reaction mechanism of P450nor, including the mechanism of the prochiral hydride transfer from NADH to the ferric heme-NO complex and identification of the chemical entity of the key spectral intermediate. The results should have gave an marked impact, to many field such as bioinorganic chemistry and P450 biochemistry.(3) Ammonia fermentation and anaerobic growth of fungi: Involvement of the assimilatory nitrate reducing system in the fungal ammonia fermentation was confirmed by characterizing the uiaD or niiA-defective mutant of the fungus Aspergillus nidulans. The denitrifying fungus F. oxysporum was shown to perform hetero-lactic acid fermentation, which is coupled to ammonia fermentation. The results together with those of denitrification show that al least a portion of soil fungi are facultative with respect to aeration, not obligate aerobe as previously thought.(4) Other subjects: Codenitrification was shown to be a biological reaction but not chemical reaction, which was supported by the results of its saturation kinetics against codenitrification substrates, and inhibition by respiration inhibitors such as cyanide. Actinomycetes are thought to be obligate aerobes, By contrast, we provided evidence for occurrence of nitrate respiration in the actinomycetes Streptomyces antibioticus and Streptomyces coelicolor. Aerobic denitrifying bacteria were isolated in order to apply them to the control of green house-effect gas emission, and their properties were characterized. Less