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（Fusarium oxysporum）等几种真菌中分离到含铜亚硝酸盐还原酶（nirK）的编码基因，首次发现真菌反硝化活性。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的细菌对口物的同源物。系统发育分析表明，近年来在许多真核微生物中发现的这些nirK基因及其同源基因均起源于单一祖先，提示它们起源于原线粒体。结果表明，尖孢霉的硝酸盐还原系统包括硝酸盐还原酶（dNaR，可能是NarGHI，普遍存在于细菌硝酸盐呼吸中）和泛素依赖性甲酸脱氢酶。与细菌系统不同，真菌dNaR系统在微有氧条件下与细胞色素氧化酶同时起作用，我们称之为混合呼吸。这些结果揭示了真菌反硝化作用在自然界中普遍存在。(2)细胞色素P450nor的反应机理：P450nor作为一氧化氮（NO）还原酶参与真菌反硝化，进行不寻常的电子转移，将2个电子从NADH直接转移到血红蛋白。通过对P450nor与NAD类似物配合物晶体结构的测定等结构和动力学分析，揭示了P450nor的详细反应机理，包括前手性氢化物从NADH向铁血红素- no配合物转移的机理以及关键光谱中间体的化学实体鉴定。研究结果对生物无机化学和P450生物化学等领域产生了重大影响。(3)氨发酵和真菌的厌氧生长：通过对真菌中性曲霉（Aspergillus nidulans）的uia或niia缺陷突变体的表征，证实了同化硝酸还原系统参与真菌氨发酵。反硝化真菌尖孢镰刀菌进行异乳酸发酵，这是偶联氨发酵。结果与反硝化的结果一起表明，至少有一部分土壤真菌在曝气方面是兼性的，而不是以前认为的专性需氧菌。(4)其他主题：co反硝化被证明是一种生物反应，而不是化学反应，其对co反硝化底物的饱和动力学结果和呼吸抑制剂（如氰化物）的抑制作用支持了这一点。放线菌被认为是专性需氧菌，相反，我们提供了硝酸盐呼吸在放线菌中发生的证据，抗生素链霉菌和冷色链霉菌。为将好氧反硝化细菌应用于温室效应气体排放的控制，分离了好氧反硝化细菌，并对其特性进行了表征。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