Nucleotide-Dependent Signal Transduction in Nitrogenase

9722937 Seefeldt固氮酶是负责生物固氮的金属酶，因此在全球氮循环中占有中心地位。固氮酶对所有底物的还原要求每个转移到底物的电子至少要有两个镁-三磷酸腺苷分子才能被水解。该研究计划的长期目标是提供对核苷酸在固氮酶反应机制中的功能的详细了解。本项目期的研究主要集中在(I)确定固氮酶铁(Fe)蛋白内核苷酸诱导的蛋白质构象变化的机制，以及这些构象变化在调节固氮酶铁蛋白与钼铁(MoFe)蛋白结合的亲和力中所起的作用，以及(Ii)确定铁蛋白-MoFe蛋白复合体中的MgATP水解物在门控和加速电子传递、促进底物还原和触发蛋白质-蛋白质解离中的功能。该方法将利用单个固氮酶组成蛋白的X射线晶体结构和固氮酶Fe蛋白-MoFe蛋白复合体的结构，结合定点突变以及生化和光谱方法来揭示固氮酶机制中每一步核苷酸的功能细节。本项目的研究结果将有助于更详细地理解核苷酸在固氮酶反应机制中的作用，并将对理解其他核苷酸偶联能量转导蛋白的机制具有更广泛的意义。还原形式的氮对地球上的所有生命都是必不可少的。在现代农业中，对减少的氮素的需求尤其有限，因为它必须以化肥的形式提供。化肥中的氮是通过需要高温和高压的化学反应来制备的。与这种化学过程相反，许多土壤细菌可以通过将大气中的氮气转化为氨来减少氮的形式，而不需要高温或压力。氮气占我们呼吸的空气的80%。这种细菌过程被称为固氮，是由一种名为固氮酶的酶催化的。固氮酶催化的氨生产目前是环境中最大的固定氮素输入，是一种非常有吸引力的替代工业制备的农业肥料。研究小组正在从分子水平上研究这一生物过程的机制，特别是对细胞能源镁-三磷酸腺苷的需求。方法是将现代基因克隆技术与生化和生物物理技术相结合，以确定固氮酶利用能源的机制。预计这些研究的结果将提供对固氮酶机制的详细了解，并将提供启动固氮酶蛋白质工程所需的基础知识。这些研究对于在农业中利用固氮酶作为一种潜在的氮源是至关重要的。

9722937 Seefeldt Nitrogenase is the metalloenzyme responsible for biological nitrogen fixation, thereby occupying a central position in the global nitrogen cycle. The reduction of all substrates by nitrogenase requires the hydrolysis of a minimum of two MgATP molecules for each electron transferred to substrates. The long range objective of this research program is to provide a detailed understanding of the functions of nucleotides in the nitrogenase reaction mechanism. The studies in the current project period focus on (i) defining the mechanisms of nucleotide induced protein conformational changes within the nitrogenase iron (Fe) protein and the roles that these conformational changes play in regulating the affinity of the nitrogenase Fe protein for binding to the molybdenum-iron (MoFe) protein and (ii) defining the functions of MgATP hydrolysis within the Fe protein-MoFe protein complex in gating and accelerating electron transfer, promoting substrate reduction, and triggering protein-protein dissociation. The approach will be to utilize the X-ray crystal structures of the individual nitrogenase component proteins and the structure of a nitrogenase Fe protein-MoFe protein complex, in conjunction with site-directed mutagenesis and biochemical and spectroscopic methods to unravel details of the functions of nucleotides at each step in the nitrogenase mechanism. It is expected that the results from this project will contribute to a detailed understanding of the functions of nucleotides in the nitrogenase reaction mechanism and will have broader implications in understanding the mechanisms of other nucleotide-coupled energy transduction proteins. Reduced forms of nitrogen are essential to all life on earth. The need for reduced nitrogen is especially limiting in modern agriculture where it must be supplied in the form of fertilizers. The nitrogen in fertilizers is prepared by a chemical reaction that requires high temperatures and pressures. In contrast to this chemical process, many soil bacteria can make reduced forms of nitrogen by conversion of atmospheric N2, a gas that constitutes 80% of the air that we breath, into ammonia without the requirements for high temperature or pressure. This bacterial process, called nitrogen fixation, is catalyzed by an enzyme called nitrogenase. Nitrogenase catalyzed ammonia production presently accounts for the largest total input of fixed nitrogen into the environment and represents a very attractive alternative to industrially prepared fertilizers for agriculture. The research team is investigating the mechanism of this biological process at the molecular level, especially the requirement for the cellular energy source MgATP. The approach will be to combine modern genetic cloning techniques with biochemical and biophysical techniques to define the mechanism of energy utilization by nitrogenase. It is anticipated that the results of these investigations will provide a detailed understanding of the mechanism of nitrogenase and will provide the foundation knowledge that will be required to initiate protein engineering of nitrogenase. Such studies will be vital to the utilization of nitrogenase as a potential source of nitrogen in agriculture.