Redox Reactions of the AdoMet Radical Enzyme Superfamily

AdoMet 自由基酶超家族的氧化还原反应

• 批准号: 9329461
• 负责人: SEAN J ELLIOTT
• 金额: $ 31.67万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329461
• 关键词: Active Sites; Address; Amino Acids; Anabolism; Behavior; Binding; Binding Proteins; Bioinformatics; Biological; Biological Models; Chemicals; Chemistry; Cobalamin; Coenzymes; Complex; Coupled; Couples; Coupling; Data; Electrochemistry; Electron Transport; Electrons; Elements; Enzymatic Biochemistry; Enzymes; Family; Family member; Film; Generations; Goals; Health; Heart; Human; Investigation; Iron; Knowledge; Measurement; Measures; Methods; Methylation; Modification; Molecular; Natural Products; Nature; Nucleic Acids; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; Play; Process; Production; Property; Prosthesis; Proteins; Protons; Reaction; Reporting; Role; S-Adenosylmethionine; Series; Site; Sulfur; Thermodynamics; Time; Ursidae Family; Work; cofactor; enzyme biosynthesis; enzyme model; experience; insight; member; mutant; oxidation; repaired; small molecule; structural biology; tool

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.

项目摘要 蛋氨酸自由基酶（ARE）超家族催化广泛的化学转化， 硫插入，异构化，甘氨酰自由基酶的活化，金属辅因子生物合成， 甲基化、氧化和去饱和。所有这些反应都是由类似的成分引发的： 由CX 3CX 2C的活性位点氨基酸特征（其结合独特的[Fe 4S 4]簇）标记， 结合所需辅因子（或辅助底物）S-腺苷甲硫氨酸（AdoMet）的结构元件。在所有 在某些情况下，战神的化学作用被认为是从活性位点还原性切割HATMet开始的 簇，产生一个5 '-dA·自由基，用于许多，许多不同的化学转化列出 以上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 已经处理了约50，000个序列，有助于将它们在许多不同生物学中的反应性分类， 涉及小分子、复杂天然产物以及蛋白质和核酸合成的作用/途径 修改. ARE超家族中的进一步复杂性可以在有多少家庭成员 另一种氧化还原辅因子，一个或两个额外的“辅助”[Fe 4S 4]簇，或钴胺素。统称 ARE超家族的催化和辅因子多样性表明，我们对ARE超家族的了解是多么的少。 分子的细节，指导战神的反应性，我们不仅不明白为什么不同的战神， 不同的反应，有关于ARE家族成员的氧化还原性质的数据很少，这是必不可少的， 了解他们如何实现他们所做的化学反应。我们建议弥补这一知识差距。我们 最近已经成功地利用晶体学表征的ARE BtrN作为模型 用于检测BtrN结合活性位点（[Fe 4S 4]Ado）和BtrN助剂的氧化还原电位的系统 簇（[Fe 4S 4]Aux），利用我们在蛋白质膜电化学方面的独特经验。在此，我们建议（i）使用 BtrN作为模型系统，可以系统评估控制氧化还原电位的因素， 质子耦合性质的活性位点，（ii）扩大我们的工作对BtrN的其他战神含有额外的FeS 簇，允许战神之间的氧化还原性质的第一次直接比较，和（iii）检查氧化还原 含钴胺素的战神的化学，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。