RESPONSES TO OXYGEN TOXICITY BY ANAEROBIC MICROORGANISMS

厌氧微生物对氧中毒的反应

• 批准号: 6520125
• 负责人: Michael W. Adams
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-07-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520125
• 关键词: Archaea; Escherichia coli; Raman spectrometry; X ray crystallography; active sites; anaerobiosis; aquatic organism; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; functional /structural genomics; infrared spectrometry; iron sulfur protein; oxidative stress; oxidoreductase; protein structure function; recombinant proteins; superoxides

Molecular O2 plays a paradoxical role in aerobic organisms in that it is both essential for energy generation as well as very toxic. Extremely reactive derivatives of O2 are produced during normal metabolism and can cause considerable damage to various cellular components, including DNA, lipids and proteins. In fact, these so-called been implicated in a wide variety of human diseases including cancer, diabetes, Alzheimer's and arthritis. The defense systems against ROS in aerobes have been well established and involve, among others, the enzyme superoxide dismutase and catalase. In contrast, the genome sequences of several anaerobes reveal that they do not contain these enzymes, which suggest that they have alternative pathways. Understanding the molecular basis of the O2 response in anaerobes has direct ramifications for the treatment of diseases caused by anaerobic pathogens. It is expected that the viability of these organisms is crucially dependent on the efficiency of their defense against ROS. Differences between anaerobes and aerobes in the pathways used to scavenger thee species might therefore be exploited as highly specific targets for drug development. The overall objective of this research is to elucidate these pathways in the hyperthermophilic anaerobe, Pyrococcus furiosus, which grows optimally at 100 degrees Centigrade. A novel non-heme iron protein termed neelaredoxin which has superoxide reductase rather than dismutase activity has been purified from this organism. The gene encoding it has been cloned and sequenced, and the crystal structure of the recombinant protein (obtained from Escherichia coli) has been solved to 1.7 A. Homologs of neelaredoxin have been found in all anaerobes examined. The P. furiosus protein is a homotetramer and contains one iron atom per subunit (of 14 kDa), which is bound to the protein in an unprecedented coordination geometry, involving four state and pre-steady state kinetics of the native protein and of mutants in which active site residues have been changed. The structural, vibrational, magnetic, electronic and redox properties of the metal center in this protein and mutants thereof, in the absence and presence of exogenous ligands, will be characterized using a range of complimentary spectroscopic techniques: electron paramagnetic resonance, optical absorption, and variable temperature magnetic circular dichroism, resonance Raman and Fourier transform infra-red. The structure of neelaredoxin, mutants and derivatives with bound exogenous ligands will be determined using crystallography. Finally, genomic and proteomic methods will be used to analyze protein patterns and gene transcripts in P. furiosus grown under various stress conditions to characterize other proteins involved in the O2 detoxification pathway.

分子O2在有氧生物中起矛盾的作用，因为它既是能量产生和非常有毒的必不可少的。 O2的极其反应性衍生物是在正常代谢过程中产生的，可能会对各种细胞成分（包括DNA，脂质和蛋白质）造成相当大的损害。实际上，这些所谓的人与多种人类疾病有关，包括癌症，糖尿病，阿尔茨海默氏症和关节炎。针对氧化杀虫剂中ROS的防御系统已得到充分确立，其中包括超氧化物歧化酶和过氧化氢酶。相反，几种厌氧菌的基因组序列表明它们不含这些酶，这表明它们具有替代途径。了解厌氧菌中O2反应的分子基础具有直接的影响，以治疗由厌氧病原体引起的疾病。 预计这些生物的生存能力至关重要地取决于他们对ROS的防御效率。因此，用于清除这种物种的途径中的厌氧菌和四氧化碳之间的差异可能被用作药物开发的高度特异性靶标。这项研究的总体目的是阐明这些途径在高疗法的厌氧菌，即富洛群虫中，该途径在100摄氏度下最佳生长。一种新型的非血红素铁蛋白称为肾上腺素，具有超氧化物还原酶而不是歧化酶活性，已从该生物体中纯化。编码它的基因已被克隆并测序，重组蛋白的晶体结构（从大肠杆菌中获得）已溶液到1.7 A. Neelaredoxin的同源物中，在所有检查的厌氧菌中都发现了。 Furiosus蛋白是一种同倍术，并包含一个亚基（14 kDa）的铁原子，该原子在前所未有的配位几何形状中与蛋白质结合，涉及四个状态和稳态的天然蛋白质和突变蛋白的稳态动力学，而突变体的突变蛋白发生了活性位点，而活性位点已改变。 The structural, vibrational, magnetic, electronic and redox properties of the metal center in this protein and mutants thereof, in the absence and presence of exogenous ligands, will be characterized using a range of complimentary spectroscopic techniques: electron paramagnetic resonance, optical absorption, and variable temperature magnetic circular dichroism, resonance Raman and Fourier transform infra-red.将使用晶体学确定具有结合的外源配体的肾氧化毒素，突变体和衍生物的结构。最后，基因组和蛋白质组学方法将用于分析在各种应力​​条件下生长的富虫的蛋白质模式和基因转录本，以表征与O2解毒途径有关的其他蛋白质。