XAS OF [NIFE]-HYDROGENASE BIOSYNTHETIC NICKEL PROTEINS

[NIFE]-氢化酶生物合成镍蛋白的 XAS

• 批准号: 7598145
• 负责人: DEBORAH B ZAMBLE
• 金额: $ 0.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598145
• 关键词: Active Sites; Anabolism; Antibiotics; Bacteria; Binding; Biotechnology; Computer Retrieval of Information on Scientific Projects Database; Data; Energy-Generating Resources; Enzymes; Escherichia coli; Funding; Gases; Grant; Helicobacter pylori; Homeostasis; Hydrogen; Hydrogenase; Individual; Institution; Iron; Knowledge; Life; Ligands; Metabolic Pathway; Metal Binding Site; Metals; Molecular; Nickel; Pathway interactions; Process; Proteins; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Sampling; Source; Spectrum Analysis; Transition Elements; United States National Institutes of Health; Work; absorption; cofactor; design; microorganism; nickel-iron hydrogenase; pathogenic bacteria; programs; research study

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. The overall aim of this research program is to determine how [NiFe]-hydrogenase enzymes are made in bacteria. These enzymes are found in many microorganisms, including Escherchia coli (E. coli) and the pathogenic bacteria Helicobacter pylori, and they are key components in the metabolic pathways that either make hydrogen gas or use it as an energy source. The enzyme has a complicated catalytic active site that contains nickel and iron bound to the protein in addition to several non-protein ligands. It is known that the biosynthesis of hydrogenases requires the coordinated activity of multiple helper proteins to deliver all of the components and assemble the metallocenter correctly. However, very little is known about the mechanisms of action of the individual proteins, how they interrelate with each other, how metal is transferred, or how metal selectivity is achieved. An understanding of this multi-step process is essential in order to realize the potential of hydrogenase enzymes for biotechnology and consumable energy applications, or to evaluate the component proteins as antibiotic targets. Furthermore, this study will contribute to our knowledge about intracellular transition metal homeostasis, a fundamental aspect of life. This experiments described in this proposal are designed to elucidate the molecular details of hydrogenase biosynthesis with a focus on the proteins that insert the nickel into the enzyme center. The proposed work is a study of the nickel insertion proteins by using X-ray absorption spectroscopy (XAS). The first experiments will provide high-resolution data about the coordination spheres of the protein metal-binding sites. We will then use XAS on multi-component samples to determine how the metal-binding sites change when the proteins interact with each other and additional cofactors. This research program will contribute to our understanding of the mechanisms of the hydrogenase biosynthetic pathway.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 该研究计划的总体目标是确定细菌中如何产生[NiFe]-氢化酶。这些酶存在于许多微生物中，包括大肠杆菌 (E. coli) 和致病菌幽门螺杆菌，它们是产生氢气或将其用作能源的代谢途径的关键组成部分。该酶具有复杂的催化活性位点，除了几个非蛋白质配体之外，还包含与蛋白质结合的镍和铁。众所周知，氢化酶的生物合成需要多种辅助蛋白的协调活性，以传递所有组分并正确组装金属中心。然而，人们对各个蛋白质的作用机制、它们如何相互关联、金属如何转移或如何实现金属选择性知之甚少。为了实现氢化酶在生物技术和消耗性能源应用中的潜力，或评估作为抗生素靶点的成分蛋白，了解这种多步骤过程至关重要。此外，这项研究将有助于我们了解细胞内过渡金属稳态，这是生命的一个基本方面。本提案中描述的实验旨在阐明氢化酶生物合成的分子细节，重点关注将镍插入酶中心的蛋白质。拟议的工作是利用 X 射线吸收光谱 (XAS) 对镍插入蛋白进行研究。第一个实验将提供有关蛋白质金属结合位点配位层的高分辨率数据。然后，我们将在多组分样品上使用 XAS 来确定当蛋白质彼此以及其他辅助因子相互作用时金属结合位点如何变化。该研究计划将有助于我们了解氢化酶生物合成途径的机制。