"MGATP Energy Transduction in Nitrogenase"

该项目的长期目标是确定MgATP水解与金属酶复合物（氮酶）中的电子转移和底物还原耦合的机制。该方法将利用最近解决的两种氮酶组分的x射线晶体结构的数据，结合位点定向诱变和改变蛋白质的生化表征来确定该反应机制中特定氨基酸的功能。氮酶铁蛋白（FeP）成分中的一系列氨基酸由于它们在22种不同生物的铁蛋白中保存，它们包含在核苷酸结合基序中以及它们在FeP的x射线结构中的位置而成为目标。本项目期间的具体目标将是阐明FeP中氨基酸参与的功能：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。已经开发出一种系统，可以通过位点定向诱变快速产生改变的FePs，并在vinelandii固氮菌中表达。改变后的蛋白质将被纯化，并进行详细的生化和生物物理表征，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、构象态分析和x射线晶体学的结构测定。初步结果已经开始确定FeP中的残基，这些残基对MgATP的结合和水解以及作为能量转导机制一部分的构象开关至关重要。预计本项目的结果将为氮酶中MgATP偶联电子传递机制提供更详细的生化理解，并将更广泛地应用于我们对其他核苷酸偶联能量转导系统的认识。最后，本研究的结果应该为启动合理的氮酶蛋白质工程以开发诸如改变核苷酸亲和力等新特性提供必要的知识基础。这个项目的长期目标是确定MgATP水解与金属酶复合物（氮酶）中的电子转移和底物还原耦合的机制。还原形式的氮对所有生物都是必不可少的。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。肥料中的氮是通过化学反应生成的，这种化学反应需要以高温和高压的形式产生大量的能量。与这种化学过程相反，许多土壤细菌可以将大气中的N2（占我们呼吸的空气的80%）转化为氨（一种农业上有用的氮源），而不需要高温或高压。这种细菌过程被称为固氮，是由固氮酶催化的。氮酶催化的氨生产占环境中固定氮的最大总投入，是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，特别是对细胞能量源MgATP的需求。我们的方法是将现代基因克隆技术与生物化学和生物物理技术相结合，以确定氮酶利用能量的机制。我们预计我们的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。这些研究对今后氮酶在农业上的利用具有一定的参考价值。＊＊＊

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. ***