Biosynthesis, Structure, and Regulation of Nitrous Oxide Reductase

一氧化二氮还原酶的生物合成、结构和调控

• 批准号: 0347871
• 负责人: David Dooley
• 金额: $ 50.03万
• 依托单位: Montana State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0347871&HistoricalAwards=false
• 关键词: Biosynthesis; Structure; Regulation; Nitrous; Oxide

This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N2O to the atmosphere, thereby contributing to ozone depletion and global warming. Hence there exist clear and direct linkages between basic research on the biology and biochemistry of denitrification and numerous issues of substantial societal interest. Specifically, studies of the structure, mechanism, and metal cluster assembly in nitrous oxide reductase will be conducted. This research will directly address questions regarding the mechanism of assembly of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Periplasmic proteins coded by the nos cluster genes nosD, L, and X (which are involved in the biosynthesis) and nitrous oxide reductase (coded by nosZ), will be purified and characterized. Investigation of cluster assembly in vitro will be conducted with purified nitrous oxide reductase and other nos proteins. Functions of the nos proteins in vivo will be probed by the generation and characterization of knockouts. Site-directed mutagenesis, together with structural, spectroscopic and kinetic methods will be used to probe the structure, bonding, and reactivity of the electron-transfer catalytic sites. Mechanistic, spectroscopic, and structural studies of the reductively-activated form of nitrous oxide reductase will be emphasized owing to the recent demonstration that this form is catalytically competent.Broader Impact: Undergraduate and graduate students will be directly involved in the research project, including students from baccalaureate colleges and groups underrepresented in science. Further, the research is conducted as part of MSU-Bozeman's IGERT program in Complex Biological Systems and therefore may contribute to institutional improvement in graduate education. Given the importance of the global nitrogen cycle and denitrification, there will be numerous opportunities (both formal and informal) to present the linkages between basic research and issues of broad interest and importance to society.

本研究主要对反硝化途径中关键酶的生物化学和结构生物学进行了研究。 固氮、同化和反硝化的环境生物学对农业生产力和水质有重大影响。 反硝化作用可能会向大气中释放N2 O，从而导致臭氧消耗和全球变暖。 因此，在反硝化的生物学和生物化学的基础研究与许多具有重大社会意义的问题之间存在着明确和直接的联系。 具体而言，将进行一氧化二氮还原酶的结构、机制和金属簇组装的研究。 这项研究将直接解决有关一氧化二氮还原酶中新型铜位点组装机制及其在催化中的作用的问题。 将纯化和表征由nos簇基因nosD、L和X（其参与生物合成）和一氧化二氮还原酶（由nosZ编码）编码的周质蛋白。 研究团簇组装在体外进行纯化的一氧化二氮还原酶和其他nos蛋白。 nos蛋白在体内的功能将通过敲除的产生和表征来探测。 定点诱变，连同结构，光谱和动力学方法将用于探测的结构，键合，和反应性的电子转移催化位点。 机械，光谱和结构研究的还原活化形式的一氧化二氮还原酶将强调由于最近的示范，这种形式是催化competitive.Broader影响：本科生和研究生将直接参与研究项目，包括学生从学士学位学院和团体在科学代表性不足。 此外，这项研究是作为MSU-博兹曼的IGERT复杂生物系统计划的一部分进行的，因此可能有助于研究生教育的机构改进。 鉴于全球氮循环和脱氮的重要性，将有许多机会（正式和非正式）介绍基础研究与社会广泛关注和重要问题之间的联系。