Redox Reactions of the AdoMet Radical Enzyme Superfamily

项目总结 ADOMet自由基酶(ARE)超家族可催化一系列跨越 硫插入、异构化、甘氨酸基酶的激活、金属辅因子的生物合成、 甲基化、氧化和去饱和化。所有这些反应都是由相似的成分启动的：ARES 由CX3CX2C的活性位点氨基酸签名(它结合唯一的[Fe4S4]簇)和 结合所需辅因子(或共底物)的结构元件S-腺苷甲基化(ADOMet)。总而言之， 在某些情况下，阿瑞斯的化学作用被认为始于活性中心对ADOMet的还原裂解 簇，产生5‘-dA·自由基，用于列出的许多不同的化学转化 上面。战神确实是一个超家族，有超过105个不同的成员，最近的生物信息学分析 已经处理了大约50,000个序列，帮助对它们在许多不同生物中的反应性进行分类 小分子、复杂天然产物、蛋白质和核酸合成的作用/途径 修改。ARE超家族内部的进一步复杂性可以从有多少家庭成员中找到 还有-另一个氧化还原辅因子，或者一个或两个额外的‘辅助’[Fe4S4]簇，或钴胺。团结在一起， ARE超家族的催化和辅助因子多样性表明我们对 指导战神反应性的分子细节，我们不仅不理解为什么不同的战神 不同的反应，关于ARE家族成员的氧化还原性质的数据很少，这是必不可少的 了解他们如何才能达到他们所做的化学反应。我们建议解决这一知识鸿沟。我们 最近已经成功地利用结晶学表征的ARE BtrN作为模型 一种检测ADOMet结合活性中心([Fe4S4]ADO)和BtrN辅助剂氧化还原电位的系统 利用我们在蛋白质膜电化学方面的独特经验，我们成功地实现了蛋白质膜电化学簇([Fe4S4]AUX)。在这里，我们建议(I)使用 BtrN作为一个模型系统，允许系统地评估是什么控制了氧化还原电位和 活性中心的质子耦合性质，(Ii)将我们在BtrN上的工作扩展到其他含有额外FeS的Are 簇，允许第一次直接比较ARE之间的氧化还原性质，以及(Iii)检查氧化还原 含钴胺ARES的化学，以进一步发展蛋白质电化学作为氧化还原的工具 ARE超家族中的酶学。

PROJECT SUMMARY The AdoMet Radical Enzyme (ARE) superfamily catalyzes a wide array of chemical transformations that span sulfur-insertion, isomerization, activation of glycyl-radical enzymes, metallo-cofactor biosynthesis, methylations, oxidations and desaturations. All of these reactions are initiated by similar components: AREs are marked by an active-site amino acid signature of CX3CX2C (which binds a unique [Fe4S4] cluster) and structural elements that bind the required co-factor (or co-substrate) S-Adenosyl-methioning (AdoMet). In all cases, the chemistry of AREs is thought to start with the reductive cleavage of AdoMet by the active site cluster, resulting in a 5'-dA· radical that is used in the many, many different chemical transformations listed above. AREs are indeed a superfamily with over 105 distinct members, and recent bioinformatics analyses have addressed ~50,000 sequences, helping to categorize their reactivity in many distinct biological roles/pathways involving the synthesis of small molecules, complex natural products, and protein and nucleic modifications. Further complexity within the ARE superfamily can be found in how many family members have yet-another redox cofactor, either one or two additional `auxiliary' [Fe4S4] clusters, or cobalamin. Together, the catalytic and cofactor diversity of the ARE superfamily articulate how very little we understand about the molecular details that guide the reactivity of AREs, we not only do not understand why different AREs do different reactions, there is little data on the redox properties of ARE family members, which is essential to understand how they can achieve the chemistry that they do. We propose to address that knowledge gap. We have have been recently successful in utilizing the crystallographically-characterized ARE BtrN as a model system for examining the redox potentials of the AdoMet-binding active site ([Fe4S4]Ado) and the BtrN auxiliary cluster ([Fe4S4]Aux) using our unique experience in protein film electrochemistry. Here, we propose to (i) use BtrN as a model system to allow for the systematic assessment of what controls the redox potentials and proton-coupled nature of the active site, (ii) expand our work on BtrN to other AREs that contain additional FeS clusters, allowing for the first direct comparison of redox properties between AREs, and (iii) examine the redox chemistry of cobalamin-containing AREs, in order to further develop protein electrochemistry as a tool for redox enzymology in the ARE superfamily.