NITROGENASE MECHANISMS AND METALLOCENTER ASSEMBLY

固氮酶机制和金属中心组件

• 批准号: 2822767
• 负责人: Lance C. Seefeldt
• 金额: $ 17.9万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2822767
• 关键词: Azotobacter vinelandii; active sites; bacterial genetics; bacterial proteins; cofactor; enzyme activity; enzyme mechanism; iron; microorganism culture; molybdenum; nitrogenase; nutrition related tag; oxidoreductase inhibitor; protein biosynthesis; site directed mutagenesis

The element nitrogen (N) is essential to all life forms on Earth and the availability of fixed nitrogen is often the limiting factor in food production. Biological nitrogen fixation, which occurs in a diverse group of microbes, represents the single largest input of fixed nitrogen in the reductive portion of the global nitrogen cycle. A molecular understanding of the enzyme responsible for biological nitrogen fixation, nitrogenase, could contribute to enhanced food production world wide, and thus to the overall health of the global population. Our goal is to contribute towards an understanding of how the two complex metallocenters of nitrogenase, the P cluster and FeMo-cofactor, are formed and function biologically. The project described herein will focus on the molecular mechanisms for P cluster and FeMo-cofactor formation, as well as the contribution of P clusters to the nitrogenase catalytic mechanism. These objectives will be accomplished through: (i) characterization of nif-specific gene products that function in the formation and insertion of FeMo-cofactor, (ii) characterization of the catalytic, spectroscopic, and kinetic properties of MoFe proteins that have been altered by site-directed mutagenesis or by the specific chemical modification of their P clusters. These experiments, which hetefore were either impossible or extremely difficult, can now be performed through the application of new purification procedures we have developed and on our ability to isolate an FeMo-cofactorless form of the MoFe protein that contains intact P clusters and can be activated to high levels of activity by FeMo-cofactor addition.

元素氮（N）对于地球上的所有生命形式至关重要 固定氮的可用性通常是食物的限制因素 生产。 生物氮固定，发生在多种多样的 一组微生物代表固定氮的最大输入 在全球氮循环的还原部分中。分子 了解负责生物氮的酶 固定酶，氮酶可能有助于增强食品生产 全球范围内，从而达到全球人口的整体健康状况。我们的 目标是有助于理解两者如何复杂 氮酶的金属中心，P簇和FEAM-CACTOR，是 形成并在生物学上发挥功能。 本文描述的项目将 专注于P簇和FEAM-COFACTOR的分子机制 形成以及P簇对氮酶的贡献 催化机制。 这些目标将通过：（i）实现 NIF特异性基因产物的表征 Femo-Cofactor的形成和插入，（II）表征 MOFE蛋白的催化，光谱和动力学特性 已通过位置定向的诱变改变或特定 其P簇的化学修饰。 这些实验，这些实验 以前是不可能的，要么极难，现在可以 通过应用新的纯化程序执行 开发并鉴于我们分离出一种无FEATO-COFACTOR形式的能力 包含完整p簇的MOFE蛋白，可以激活至 添加女性化合物的高水平活性。