"MGATP Energy Transduction in Nitrogenase"

Seefeldt 9315835本项目的长期目标是确定MgATP水解与金属酶复合物（固氮酶）中的电子转移和底物还原耦合的机制。 该方法将使用来自最近解决的固氮酶组分的X射线晶体结构的数据，结合定点诱变和改变的蛋白质的生物化学表征来定义该反应机制中特定氨基酸的功能。 固氮酶的铁蛋白（FeP）组分内的一系列氨基酸已被靶向，因为它们在来自22种不同生物体的铁蛋白之间的保守性，它们在核苷酸结合基序中的包含以及它们在FeP的X射线结构中的位置。 本项目期间的具体目标是阐明FeP中氨基酸的功能，这些功能涉及：1）核苷酸磷酸结合和水解，2）腺嘌呤识别，3）组分对接和电子转移以及4）MgATP诱导的构象变化。 已经开发了一种系统，该系统允许通过定点诱变在棕色固氮菌中表达来快速产生改变的FePs。 将纯化改变的蛋白质并进行详细的生物化学和生物物理表征，以确定原始氨基酸的功能。 这些表征研究将包括稳态和预稳态动力学研究、构象状态分析和X射线晶体学结构测定。 初步结果已经开始定义的FeP中的残基是必不可少的MgATP的结合和水解和构象开关的能量转导机制的一部分。 预计该项目的结果将提供固氮酶中MgATP偶联电子转移机制的更详细的生物化学理解，并将对我们对其他核苷酸偶联能量转导系统的知识具有更广泛的应用。 最后，从这项研究的结果应该提供一个基础的知识，需要启动合理的蛋白质工程固氮酶开发新的性能，如改变核苷酸亲和力。 本项目的长期目标是确定MgATP水解与金属酶复合物固氮酶中的电子转移和底物还原耦合的机制。 还原态氮对所有生物体都是必不可少的。 在必须以肥料形式提供氮的现代农业中，对减少氮的需求特别有限。 肥料中的氮是通过化学反应制备的，需要大量的高温和高压形式的能量。 与这种化学过程相反，许多土壤细菌可以将大气中的N2（占我们呼吸空气的80%）转化为氨（农业上有用的氮源），而不需要高温或高压。 这种细菌过程称为固氮，由固氮酶催化。 固氮酶催化的氨生产占环境中固定氮的最大总输入，并且代表了一种非常有吸引力的替代工业制备的农业肥料。 我们正在分子水平上研究这一生物过程的机制，特别是对细胞能量来源MgATP的需求。 我们的方法将是将联合收割机现代基因克隆技术与生物化学和生物物理技术相结合，以确定固氮酶的能量利用机制。 我们预计，我们的调查结果将提供一个详细的了解固氮酶的机制，并将提供基础知识，将需要启动固氮酶的蛋白质工程。 这些研究对今后固氮酶在农业上的应用具有重要意义。 ***

Seefeldt 9315835 The long range objective of this project is to define the mechanism by which MgATP hydrolysis is coupled to electron transfer and substrate reduction in the metalloenzyme complex, nitrogenase. The approach will be to use date from the recently solved x-ray crystal structures of both nitrogenase components coupled with site- directed mutagenesis and biochemical characterization of altered proteins to define the functions of specific amino acids in the mechanism of this reaction. A series of amino acids within the iron protein (FeP) components of nitrogenase have been targeted as a result of their conservation among iron proteins from 22 different organisms, their inclusion in nucleotide binding motifs and by their location in the x-ray structure of the FeP. The specific objectives for this project period will be to elucidate the function of amino acids within the FeP involved in: 1) nucleotide phosphate binding and hydrolysis, 2) adenine recognition, 3) component docking and electron transfer and 4) the MgATP induced conformational change. A system has been developed that allows the rapid generation of altered FePs by site-directed mutagenesis with expression in Azotobacter vinelandii. The altered proteins will be purified and subjected to detailed biochemical and biophysical characterizations to determine the function of the original amino acid. These characterization studies will include steady state and pre-steady state kinetic studies, analysis of conformational states, and structure determination by x-ray crystallography. Preliminary results have begun to define residues in the FeP that are essential to MgATP binding and hydrolysis and to the conformational switch that is part of the energy transduction mechanism. It is expected that the results from this project will provide a more detailed biochemical understanding of the MgATP coupled electron transfer mechanism in nitrogenase and will have broader applications to our knowledge of other nucleotide coupled energy transduction systems. Finally, the results from this study should provide a foundation of knowledge required to initiate rational protein engineering of nitrogenase to develop new properties such as altered nucleotide affinities. %%% The long range objective of this project is to define the mechanism by which MgATP hydrolysis is coupled to electron transfer and substrate reduction in the metalloenzyme complex, nitrogenase. Reduced forms of nitrogen are essential to all living organisms. 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 large amounts of energy in the form of high temperatures and pressures. In contrast to this chemical process, many soil bacteria can convert atmospheric N2, a gas that constitutes 80% of the air that we breath, into ammonia, an agriculturally useful nitrogen source, without the requirements for high temperature or pressure. This bacterial process, called nitrogen fixation, is catalyzed by the enzyme nitrogenase. Nitrogenase catalyzed ammonia production accounts for the largest total input of fixed nitrogen into environment and represents a very attractive alternative to industrially prepared fertilizers for agriculture. We are investigating the mechanism of this biological process at the molecular level, especially the requirement for the cellular energy source MgATP. Our approach will be to combine modern genetic cloning techniques with biochemical and biophysical techniques to define the mechanism of energy utilization by nitrogenase. We anticipate that the results of our 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 could prove valuable to the future utilization of nitrogen ase in agriculture. ***