Radical SAM Enzymes: Molecular Mechanisms of Radical Initiation

Broderick，Joan B. 5R35GM131889-02 购买快速冷冻淬火/停止流的行政补充 摘要/项目概要 自由基SAM是已知的最大的酶超家族之一，其成员催化一种 在生活的各个领域都有着惊人的多样性反应。自由基SAM酶参与合成 必需的辅助因子和抗生素，DNA损伤的修复，以及复杂生物金属的组装 集群，以及许多其他反应。自由基SAM酶在人类中的存在，以及在这两个 有益微生物和致病微生物对于了解它们的基本特性具有重要意义 和机制。拟议中的研究将发展对自由基SAM机制的批判性理解， 特别是自由基引发过程，该过程对于该大而多样的超家族中的所有酶都是共同的。 研究工作将集中在捕获自由基中间体使用快速冷冻淬火，低温还原， 退火和光解方法。然后，这些中间体将通过使用 电子顺磁共振和电子-核双谱 共振和结构方法，如X射线晶体学。拟议的补充将允许 购买所需的停流混合/快速冷冻淬火和紫外可见检测能力， 进行这些实验。

Broderick, Joan B. 5R35GM131889-02 Administrative Supplement for Purchase of a Rapid Freeze Quench / Stopped Flow Abstract/Summary of Project Radical SAM is one of the largest enzyme superfamilies known, with its members catalyzing a remarkable diversity of reactions in all domains of life. Radical SAM enzymes are involved in the synthesis of essential cofactors and antibiotics, repair of DNA damage, and the assembly of complex biological metal clusters, among many other reactions. The presence of radical SAM enzymes in humans as well as in both beneficial and pathogenic microbes lends high significance to understanding their fundamental properties and mechanisms. The proposed research will develop a critical understanding of radical SAM mechanisms, in particular the radical initiation process common to all enzymes in this large and diverse superfamily. Research efforts will focus on trapping radical intermediates using rapid freeze-quench, cryoreduction, annealing, and photolysis approaches. These intermediates will then be characterized by using spectroscopic approaches such as electron paramagnetic resonance and electron-nuclear double resonance, and structural approaches such as X-ray crystallography. The proposed supplement will allow the purchase of needed stopped-flow mixing/ rapid-freeze-quench and UV-visible detection capability to carry out these experiments..