Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

项目摘要。 自由基S-腺苷-L-蛋氨酸(SAM)超家族酶存在于所有的生命王国和 使用铁-硫团簇催化广泛的反应。这些不同的反应出人意料地引发了 通过一个统一的过程，该过程开始于共底物/共因子SAM与铁-硫簇的结合。 用铁-硫簇还原裂解SAM，生成反应性自由基中间体a 5- 脱氧腺苷自由基。本种是一个强大的氢原子抽取器，并被广泛接受为该种 负责自由基SAM酶的反应。这种中间体的产生还没有完全完成 因为它在反应途径中的存在直到最近才从间接检测中推断出来 方法：研究方法。基于最近对这种中间体的直接观察，在没有光解的情况下使用 底物，以及通过使用非原生底物，这个项目的目的是捕捉和表征 在催化相关条件下的自由基中间体，并连接发现的有机金属中间体 是催化这种强有力的有机自由基中间体的中心。将使用几种方法来调查 这些中间体的存在会导致底物自由基的产生。A肽 模拟研究充分的自由基SAM酶的大分子底物将被用来捕获5- 脱氧腺苷自由基是一个稳定的物种。其次，另一种研究得很好的SAM酶，其反应可以是 在特定条件下放慢反应速度将使我们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、快速冷冻淬火、低温还原和顺序退火法。 技巧。中间体的表征将使用电子顺磁共振和 电子核双共振谱实验，以及X射线结晶学。SAM的使用 同位素将干扰潜在中间体的光谱信号，从而进行结构鉴定。 瞬变中间体。拟议研究的目标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 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，并有可能在药学上 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始独立的研究生涯。此字段由多个方面组成 对无机化学、分子生物学、物理学和生物化学产生深远影响 生理学、药理学和毒理学。为了更好地为这一领域做出贡献，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这份奖学金将使我获得 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 机制和光谱技能。蒙大拿州立大学有一群令人难以置信的科学家 这可以扩大我对生物无机化学方方面面的理解。

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.