RESPONSES TO OXYGEN TOXICITY BY ANAEROBIC MICROORGANISMS

厌氧微生物对氧中毒的反应

基本信息

批准号：
6849573
负责人：
Michael W. Adams
金额：
$ 6.81万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
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/6849573
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

项目摘要

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.

O2 分子在需氧生物体中扮演着矛盾的角色，因为它既是能量产生所必需的，又具有剧毒。 O2 的活性极高的衍生物是在正常代谢过程中产生的，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。事实上，这些所谓的疾病与多种人类疾病有关，包括癌症、糖尿病、阿尔茨海默病和关节炎。需氧菌中针对 ROS 的防御系统已经非常完善，其中包括超氧化物歧化酶和过氧化氢酶。相比之下，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 O2 反应的分子基础对于治疗厌氧病原体引起的疾病具有直接影响。预计这些生物体的生存能力很大程度上取决于它们防御活性氧的效率。因此，厌氧菌和需氧菌在清除这些物种的途径上的差异可能被用作药物开发的高度特异性靶标。这项研究的总体目标是阐明超嗜热厌氧菌激烈火球菌中的这些途径，该微生物在 100 摄氏度下生长最佳。一种新型非血红素铁蛋白，称为 Neelaredoxin，具有超氧化物还原酶活性，而不是歧化酶活性，已从该生物体中纯化出来。编码它的基因已被克隆和测序，重组蛋白（从大肠杆菌获得）的晶体结构已解析为1.7 A。在所有检查的厌氧菌中都发现了neelaredoxin的同源物。 P. Furiosus 蛋白是一种同四聚体，每个亚基（14 kDa）含有一个铁原子，它以前所未有的配位几何结构与蛋白质结合，涉及天然蛋白质和突变体的四种状态和前稳态动力学。哪些活性位点残基已被改变。在存在和不存在外源配体的情况下，该蛋白质及其突变体中金属中心的结构、振动、磁性、电子和氧化还原特性将使用一系列互补的光谱技术进行表征：电子顺磁共振、光吸收和变温磁圆二色性、共振拉曼和傅立叶变换红外。将使用晶体学测定具有结合的外源配体的近氧还蛋白、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胁迫条件下生长的 P. Furiosus 的蛋白质模式和基因转录本，以表征参与 O2 解毒途径的其他蛋白质。