Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

项目摘要。 自由基 S-腺苷-L-甲硫氨酸 (SAM) 酶超家族存在于所有生命领域，并且 使用铁硫簇催化广泛的反应。这些不同的反应是令人惊讶地引发的 通过一个统一的过程，该过程从辅助底物/辅助因子 SAM 与铁硫簇的结合开始。 铁硫簇对 SAM 进行还原裂解，生成活性自由基中间体，即 5- 脱氧腺苷自由基。该物种是一种有效的氢原子提取剂，被广泛认为是 负责自由基 SAM 酶的反应性。该中间体的生成尚未完全 由于直到最近才从间接检测中推断出它在反应途径中的存在，因此已被理解 方法。基于最近在没有光解的情况下直接观察到这种中间体 基质，以及通过使用非天然基质，该项目旨在捕获和表征 在催化相关条件下生成自由基中间体，并连接发现的有机金属中间体 对于这种有效的有机自由基中间体的催化至关重要。将采用多种方法来探究 反应途径中这些中间体的存在导致底物自由基的产生。一种肽 经过充分研究的自由基 SAM 酶的大分子底物模拟物将用于捕获 5- 脱氧腺苷自由基作为稳定的物种。其次，另一种经过充分研究的 SAM 酶，其反应可以是 在特定条件下减慢将允许有机会研究反应性自由基中间体。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 同位素体会扰乱潜在中间体的光谱信号，从而实现结构识别 的瞬态中间体。拟议研究的目的是将拟议的催化中间体连接起来 发挥作用并提供电子和几何细节，以帮助阐明自由基引发机制 通过自由基 SAM 酶。了解这种生化反应将有助于这些酶的应用 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，并具有药物开发的潜力 靶向人类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生涯。该字段由切面组成 无机化学、分子生物学、物理学和生物化学的学科，可以对 生理学、药理学和毒理学。为了最好地能够为这个领域做出贡献，我的培训必须 扩展到包括生物科学的各个方面。布罗德里克实验室的这次奖学金将使我获得 蛋白质表达和纯化方面的经验以及无机反应阐明方面的经验 机制和光谱技能。蒙大拿州立大学有一个令人难以置信的科学家社区 这可以扩大我对生物无机化学各个方面的理解。

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.