Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

探究自由基 SAM 酶化学中统一的自由基生成步骤

• 批准号: 10447575
• 负责人: Maike Nicole Lundahl
• 金额: $ 6.76万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447575
• 关键词: Anabolism; Antibiotics; Binding; Biochemical Reaction; Biochemistry; Bioinorganic Chemistry; Biological Sciences; Biotechnology; Catalysis; Chemistry; Communities; Competence; Complex; DNA Repair; Development; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Enzyme Reactivation; Enzymes; Fellowship; Freezing; Future; Generations; Glycine; Health; Human; Hydrogen; Inorganic Chemistry; Iron; Kinetics; Laboratories; Life; Link; Methionine; Molecular Biology; Montana; Nature; Pathway interactions; Peptides; Pharmacologic Substance; Pharmacology and Toxicology; Physics; Physiology; Play; Positioning Attribute; Process; Property; Reaction; Reporting; Research; Ribonucleotide Reductase; Role; S-Adenosylmethionine; Scientist; Signal Transduction; Site; Sulfur; System; Techniques; Temperature; Training; Universities; Variant; Vitamins; X-Ray Crystallography; analog; beneficial microorganism; career; detection method; experience; experimental study; formate acetyltransferase activating enzyme; human pathogen; insight; macromolecule; member; metal complex; metalloenzyme; non-Native; pathogenic microbe; peptidomimetics; photolysis; polypeptide; protein purification; skills; therapy design; tool

Project Summary. The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes are found in all kingdoms of life and use an iron-sulfur cluster to catalyze a broad range of reactions. These diverse reactions are surprisingly initiated by a unified process which begins with the binding of co-substrate/co-factor, SAM, to an iron-sulfur cluster. Reductive cleavage of SAM by the iron-sulfur cluster generates the reactive radical intermediate, a 5- deoxyadenosyl radical. This species is a potent hydrogen atom abstractor and is widely accepted as the species responsible for radical SAM enzymes’ reactivity. The generation of this intermediate has yet to be fully understood since its presence in the reaction pathway has until recently only been inferred from indirect detection methods. Building on the recent direct observation of this intermediate using photolysis in the absence of substrate, as well as through the use non-native substrates, this project aims to capture and characterize the radical intermediate under catalytically relevant conditions, and to link an organometallic intermediate found to be central to catalysis to this potent organic radical intermediate. Several approaches will be used to probe the presence of these intermediates in the reaction pathway resulting in substrate radical generation. A peptide mimic of the macromolecular substrate of a well-studied radical SAM enzyme will be used to capture the 5- deoxyadenosyl radical as a stable species. Secondly, another well-studied SAM enzyme whose reaction can be slowed under particular conditions will allow for the opportunity to study the reactive radical intermediates. These two enzyme systems will be used with photolysis, rapid freeze quench, cryoreduction, and sequential annealing techniques. Characterization of intermediates will be carried out using electron paramagnetic resonance and electron nuclear double resonance spectroscopic experiments, as well as X-ray crystallography. The use of SAM isotopologues will perturb the spectroscopic signals of potential intermediates, allowing structural identification of transient intermediates. The objective of the proposed research is to connect proposed catalytic intermediates to function and provide electronic and geometric details to aid in elucidation of the mechanism of radical initiation by radical SAM enzymes. Understanding this biochemical reaction will allow for the application of these enzymes as a powerful biocatalyts capable of numerous challenging reactions and for the potential to pharmaceutically target radical SAM enzymes in humans and pathogens. I aim to launch an independent research career in bioinorganic chemistry. This field is made up of facets of inorganic chemistry, molecular biology, physics and biochemistry and can have profound impacts on physiology, pharmacology and toxicology. In order to best be able to contribute to this field, my training must expand to include aspects of biological sciences. This fellowship in the Broderick laboratory will allow me to gain experience in the expression and purification of proteins and build on experience in elucidation inorganic reaction mechanisms and spectroscopic skills. There is an incredible community of scientists at Montana State University that can expand my understanding of all facets of bioinorganic chemistry.

项目摘要。 自由基S-腺苷-L-甲硫氨酸（SAM）超家族酶存在于所有生命界， 使用铁硫簇催化广泛的反应。令人惊讶的是，这些不同的反应 通过一个统一的过程，该过程开始于共底物/辅因子SAM与铁硫簇的结合。 铁硫簇合物对SAM的还原裂解产生反应性自由基中间体，即5 π- 脱氧腺苷自由基这一物种是一个强大的氢原子提取器，并被广泛接受为物种 负责自由基SAM酶的反应性。这种中间体的产生还没有完全 之所以理解，是因为直到最近它在反应途径中的存在才通过间接检测来推断 方法.基于最近在没有光的情况下使用光解对这种中间体的直接观察， 基板，以及通过使用非原生基板，该项目的目的是捕捉和表征 自由基中间体在催化相关的条件下，并连接有机金属中间体发现， 是催化这种有效的有机自由基中间体的核心。将使用几种方法来探测 这些中间体在反应途径中的存在导致底物自由基的产生。的肽 将使用一种充分研究的自由基SAM酶的大分子底物的模拟物来捕获5 ′- 脱氧腺苷自由基作为一个稳定的物种。其次，另一种研究充分的SAM酶，其反应可以是 在特定条件下减慢将允许有机会研究活性自由基中间体。这些 两种酶系统将用于光解、快速冷冻淬灭、冷冻还原和顺序退火 技术.中间体的表征将使用电子顺磁共振和 电子核双共振光谱实验，以及X射线晶体学。SAM的使用 同位素体将干扰潜在中间体的光谱信号，允许结构鉴定 瞬时中间体的。拟议研究的目的是连接拟议的催化中间体 发挥作用并提供电子和几何细节，以帮助阐明自由基引发的机制 自由基SAM酶的作用。了解这种生化反应将允许这些酶的应用 作为一种强大的生物催化剂，能够进行许多具有挑战性的反应， 靶向人体和病原体中的自由基SAM酶。 我的目标是在生物无机化学领域开展独立的研究工作。此字段由面组成 无机化学、分子生物学、物理学和生物化学的研究，并可能对 生理学、药理学和毒理学。为了能够更好地为这个领域做出贡献，我的培训必须 扩大到包括生物科学的各个方面。布罗德里克实验室的奖学金能让我 在蛋白质的表达和纯化方面的经验，以及在阐明无机反应方面的经验 机制和光谱学技能。在蒙大拿州立大学有一个令人难以置信的科学家社区 可以扩展我对生物无机化学各个方面的理解。