HUMAN ETHE1: ACTIVITY AND CATALYTIC MECHANISM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 微量元素对所有活着的有机体都是必不可少的。同时，细胞内游离金属离子的浓度很低，因为自由氧化还原活性金属会产生自由基，从而导致细胞损伤。其他的，如钙和锌，也参与了信号传递过程。这意味着有必要进行严格的监管。在哺乳动物中，调节功能的障碍会导致严重的疾病。如果金属含量过高或过低，细菌也会遇到类似的问题。鉴于哺乳动物和细菌的微量元素调控是基于不同的蛋白质网络，细菌金属调节剂作为潜在的药物靶点引起了人们的极大关注。在过去的几年里，我们已经建立了对细菌金属调控的研究。病原体，如结核分枝杆菌，为了在人体内生存，必须与铁的隔离作斗争。铁的代谢是通过控制与铁的吸收、运输和储存有关的基因的转录来调节的。在结核分枝杆菌中，铁摄取调节因子A(FURA)被Fe2激活，与其靶DNA序列特异性结合，从而抑制下游基因。在同源病例中，这些基因包括毒力因子和细菌维持铁稳态所需的几种蛋白质。通过X射线吸收光谱，我们将确定蛋白质的金属结合部位。这使得能够区分两个结构和功能不同的FurAMtb金属结合位点，并提供了详细的描述以及对它们的定性和定量表征。在这里，我们建议对结核分枝杆菌铁摄取调节因子Fura进行X射线吸收光谱测量，以便在没有和存在新的先导化合物的情况下进一步表征这一重要的基因产物。为了进行比较，我们将包括来自氧化亚铁硫杆菌的Fur_AF，这是一种生活在PH22中的革兰氏阴性细菌，含有高浓度的可溶性铁和铁离子。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Trace elements are essential for all living organisms. At the same time in cells the concentration of free metal ions is very low, because free redox-active metals can generate radicals, which can lead to cell damage. Others, such as Ca and Zn are participating in signaling processes. This implies the need for tight regulation. In mammals dysfunction of the regulation causes severe diseases. Bacteria would suffer from similar problems in case of too high or too low metal contents. Given the fact that mammalian and bacterial trace element regulation is based on different protein networks, the bacterial metal regulators have attracted considerable attention as potential drug targets. During the past years we have established our research on bacterial metal regulation. Pathogens, such as M. tuberculosis have to contend with iron sequestration in order to survive in the human body. Iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In M. tuberculosis the ferric uptake regulator A (FurA) is activated by Fe2+ to bind specifically to its target DNA sequence thereby repressing the downstream genes. These genes include in homologous cases virulence factors and several proteins required by the bacterium for iron homeostasis. By x-ray absorption spectroscopy we will determine the metal binding sites of the protein. This allows distinguishing between two structurally and functionally distinct FurAMtb metal binding sites and provides a meticulous description plus a qualitative and quantitative characterization of them. Here we propose x-ray absorption spectroscopy measurements on the Mycobacterium tuberculosis iron uptake regulator FurA to further characterize this important gene product in the absence and presence of a new lead compound. For comparison we will include Fur_AF from Acidithiobacillus ferrooxidans, a Gram-negative bacterium living at pH2 2 in high concentrations of soluble ferrous and ferric iron.