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

真核和原核细胞多重异化氮代谢的分子机制

基本信息

批准号：
14104005
负责人：
SHOUN Hirofumi
金额：
$ 72.97万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2007
项目状态：
已结题

项目摘要

(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

(1)真菌反硝化系统的分子机制：编码含铜亚硝酸还原酶(nirK)的基因是从尖孢镰刀菌(Fusarium oxysporum)等多种真菌中分离得到的，其中NirK蛋白是之前从尖孢镰刀菌(Fusarium oxysporum)中首次发现的真菌反硝化活性。被证明是 nirK 基因的产物，这是从真核生物中首次分离出参与其中的细菌对应物的直系同源物。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基因及其同源基因均起源于单一祖先，表明它们源自尖镰孢的硝酸盐还原系统。包含硝酸还原酶（dNaR，可能是 NarGHI，普遍存在于细菌硝酸盐呼吸中）和泛醌依赖性甲酸与细菌系统不同，真菌 dNaR 系统在微需氧条件下与细胞色素氧化酶同时发挥作用，我们将其称为混合呼吸。（2）反应机制。细胞色素 P450nor 的：P450nor 作为一氧化氮 (NO) 还原酶参与真菌反硝化，执行不寻常的电子转移，直接2 个电子从 NADH 转移到血红素蛋白结构和动力学分析，例如确定 P450nor 与 NAD 类似物复合物的晶体结构，揭示了 P450nor 的详细反应机制，包括前手性氢化物从 NADH 转移到血红素蛋白的机制。铁血红素-NO复合物和关键光谱中间体的化学实体的鉴定该结果应该对生物无机化学和化学等许多领域产生显着影响。 P450生物化学。(3)氨发酵和真菌的厌氧生长：通过表征真菌构巢曲霉(Aspergillus nidulans)的uiaD或niiA缺陷突变体，证实了同化硝酸盐还原系统参与真菌氨发酵。显示可以进行异乳酸发酵，该发酵与氨发酵相结合。与反硝化作用的结果一起表明，至少有一部分土壤真菌是兼性通气的，而不是像以前认为的专性需氧菌。（4）其他课题：硝化作用被证明是一种生物反应，而不是化学反应，这使得微生物的反应更加容易。其针对共硝化底物的饱和动力学结果支持了这一点，并且放线菌等呼吸抑制剂的抑制被认为是专性需氧微生物。我们提供了放线菌中抗生素链霉菌和天蓝色链霉菌发生硝酸盐呼吸的证据，以便将它们应用于温室效应气体排放的控制，并对其特性进行了表征。