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.

氮元素 (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品的限制因素 生产。 生物固氮作用发生在多种情况 微生物群，代表固定氮的单一最大输入 在全球氮循环的还原部分。一个分子 了解负责生物氮的酶 固氮酶的固定作用可能有助于提高粮食产量 世界范围内，从而影响全球人口的整体健康。我们的 目标是帮助理解这两个复杂的 固氮酶的金属中心、P 簇和 FeMo 辅因子是 形成并发挥生物学功能。 本文描述的项目将 关注P簇和FeMo辅因子的分子机制 形成以及 P 簇对固氮酶的贡献 催化机制。 这些目标将通过以下方式实现：(i) nif 特异性基因产物的表征，该产物在 FeMo 辅因子的形成和插入，(ii) 的表征 MoFe 蛋白质的催化、光谱和动力学特性 已通过定点诱变或特定的 他们的P簇的化学修饰。 这些实验， 以前要么是不可能的，要么是极其困难的，现在可以 通过应用新的纯化程序进行，我们有 开发并基于我们分离 FeMo 无辅因子形式的能力 MoFe 蛋白含有完整的 P 簇，可被激活 添加 FeMo 辅因子可提高活性水平。