Biosynthesis, Structure, Function, and Regulation of NitrousOxide Reductase

一氧化二氮还原酶的生物合成、结构、功能和调控

• 批准号: 9723715
• 负责人: David Dooley
• 金额: $ 27万
• 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: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723715&HistoricalAwards=false
• 关键词: Biosynthesis; Structure; Function; Regulation; NitrousOxide

9723715 Dooley The biochemistry, biosynthesis, structures, reactivity, and function of nitrous oxide reductase, the terminal enzyme in bacterial denitrification, is being investigated. Denitrification is a key component of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The ecology involving nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N20 to the atmosphere, where it may contribute to ozone depletion and global warming. This research addresses several outstanding issues in the biochemistry of denitrification, in the bacterium Achromobacter cycloclastes, with the goal of a thorough molecular understanding of this pathway. The roles of the proteins encoded by the genes nosD, F,Y,L,X, which are (or may be) required for the biosynthesis of nitrous oxide reductase, are being examined. The general strategy is to clone and over-express the soluble, periplasmic nos-coded proteins, which may be involved in the biosynthesis of the catalytic site of nitrous oxide reductase, thereby insuring sufficient material for detailed biophysical and functional studies. Site-directed mutagenesis, together with variable -temperature absorption, CD, MCD, EPR and resonance Raman spectroscopy will be used to probe the nature of the catalytic site in nitrous oxide reductase (thought to be the Cuz site), and the mechanism of reduction of nitrous oxide, including the interactions with physiological electron donors. Concerted efforts are underway to develop rapid, generally applicable schemes to purify nitrous oxide reductase, with the objective of obtaining crystals of nitrous oxide reductase suitable for x-ray diffraction analysis. Results of these experiments should be applicable to major themes in modern biochemistry such as metalloenzyme structure and function; metal ion metabolism and the biosynthesis of metalloproteins; and the recognition and acti vation of kinetically-inert small molecules. This research project seeks to understand how bacteria in soil and water convert nitrate, an important source of nitrogen for plants, into nitrogen gas, which is released to the atmosphere. A substantial fraction of the fertilizers applied to crops are wasted as a result of this conversion. The organisms responsible contain an enzyme known as nitrous oxide reductase, which plays a critical role in the overall process. A major goal of the project is to understand how this enzyme is made, its structure, and exactly how it functions. Understanding how bacteria convert nitrate to nitrogen gas could eventually lead to effective controls of this process, thereby resulting in substantial savings to farmers. It might also be possible to turn the tables and exploit this process to safely remove nitrate from drinking-water supplies, where it can be a problem.

氧化亚氮还原酶是细菌反硝化的终端酶，目前正在研究其生物化学、生物合成、结构、反应性和功能。反硝化是全球氮循环的关键组成部分，是平衡循环的途径，将固定的氮返回到大气中。包括固氮、同化和反硝化在内的生态系统对农业生产力和水质有重大影响。反硝化可能向大气释放N20，在那里它可能导致臭氧消耗和全球变暖。本研究解决了反硝化生物化学中的几个突出问题，在细菌无色杆菌环裂菌中，目标是对这一途径进行彻底的分子理解。由nosD， F，Y，L，X基因编码的蛋白质是（或可能是）氧化亚氮还原酶生物合成所必需的，其作用正在研究中。一般策略是克隆和过表达可溶的、质周无编码的蛋白质，这些蛋白质可能参与氧化亚氮还原酶催化位点的生物合成，从而为详细的生物物理和功能研究提供足够的材料。位点定向诱变，结合变温吸收、CD、MCD、EPR和共振拉曼光谱，将探讨氧化亚氮还原酶的催化位点（被认为是Cuz位点）的性质，以及氧化亚氮还原的机制，包括与生理电子供体的相互作用。为了获得适合x射线衍射分析的氧化亚氮还原酶晶体，人们正在共同努力开发快速、普遍适用的氧化亚氮还原酶纯化方案。这些实验结果应适用于金属酶的结构和功能等现代生物化学的重大课题；金属离子代谢与金属蛋白的生物合成以及对惰性小分子的识别和激活。该研究项目旨在了解土壤和水中的细菌如何将硝酸盐（植物氮的重要来源）转化为释放到大气中的氮气。由于这种转化，施用在作物上的化肥有很大一部分被浪费了。负责的生物体含有一种被称为氧化亚氮还原酶的酶，它在整个过程中起着关键作用。该项目的一个主要目标是了解这种酶是如何制造的，它的结构，以及它的确切功能。了解细菌如何将硝酸盐转化为氮气可能最终导致对这一过程的有效控制，从而为农民节省大量资金。也有可能扭转局面，利用这一过程安全地去除饮用水供应中的硝酸盐，这可能是一个问题。