Nucleotide-Dependent Signal Transduction in Nitrogenase

Seefeldt氮酶是负责生物固氮的金属酶，因此在全球氮循环中占据中心位置。所有底物的氮酶还原需要水解至少两个MgATP分子为每个电子转移到底物。该研究计划的长期目标是提供对核苷酸在氮酶反应机制中的功能的详细了解。当前项目期的研究重点是(i)确定核苷酸诱导的氮化酶铁（Fe）蛋白内蛋白质构象变化的机制，以及这些构象变化在调节氮化酶铁蛋白与钼铁（MoFe）蛋白结合的亲和力中所起的作用；（ii）确定Fe蛋白-MoFe蛋白复合物内MgATP水解在控制和加速电子转移中的作用。促进底物还原，引发蛋白质解离。该方法将利用单个氮酶成分蛋白的x射线晶体结构和氮酶Fe蛋白- mofe蛋白复合物的结构，结合定点诱变和生化和光谱方法，揭示核苷酸在氮酶机制中每一步的功能细节。预计本项目的结果将有助于详细了解核苷酸在氮酶反应机制中的功能，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。现代农业对还原性氮的需求尤其有限，因为还原性氮必须以肥料的形式提供。肥料中的氮是通过需要高温高压的化学反应产生的。与这种化学过程相反，许多土壤细菌可以通过将大气中的N2（一种占我们呼吸空气80%的气体）转化为氨来制造还原型氮，而不需要高温或高压。这种细菌过程被称为固氮，是由一种叫做氮酶的酶催化的。目前，氮酶催化的氨生产占环境中固定氮的最大总投入，是工业制备的农业肥料的一个非常有吸引力的替代方案。研究小组正在分子水平上研究这一生物过程的机制，特别是对细胞能量源MgATP的需求。将现代基因克隆技术与生物化学和生物物理技术相结合，确定氮酶利用能量的机制。预计这些研究结果将提供对氮酶机制的详细了解，并将为启动氮酶蛋白质工程所需的基础知识。这些研究将对利用氮酶作为农业氮的潜在来源至关重要。

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.