Radical Mechanisms of Iron-Sulfur Proteins

铁硫蛋白的自由基机制

• 批准号: 9277146
• 负责人: SQUIRE J. BOOKER
• 金额: $ 29.88万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9277146
• 关键词: Aerobic Bacteria; Amaze; Anabolism; Anaerobic Bacteria; Antibiotic Resistance; Antibiotics; Carbon; Cleaved cell; DNA Repair; DNA biosynthesis; Disease; Dissociation; Enzymes; Free Radicals; Genome; Health; Human; Hydrogen; Hydrogen Bonding; Individual; Iron; Iron-Sulfur Proteins; Laboratories; Methods; Methylation; Methyltransferase; Modification; Pathway interactions; Phosphorus; Play; Process; Proteins; Reaction; Resistance; Ribosomes; Role; S-Adenosylmethionine; Source; Sulfur; Thioctic Acid; Transfer RNA; Transition Elements; Viral; Work; cobamamide; cofactor; member; novel

PROJECT SUMMARY Elaborations of unactivated carbon centers are among the most demanding reactions that enzymes catalyze. These reactions generally involve radical intermediates, often produced by strategic abstraction of substrate hydrogen atoms (H·). A prevalent strategy to cleave C–H bonds possessing homolytic bond-dissociation energies (BDEs) in excess of 95 kcal/mol involves intermediates derived from the reaction of O2 with transition metal cofactors. A distinct strategy, predominant in the anaerobic world and still important in aerobes, employs a 5'-deoxyadenosyl 5'-radical as the H· abstractor. This radical is generated via the homolysis of adenosylcobalamin (AdoCbl) or the reductive cleavage of S-adenosylmethionine (SAM). Those enzymes employing SAM to catalyze radical-dependent reactions belong to the so-called radical SAM (RS) superfamily, which contains almost 114,000 individual sequences that encompass at least 65 distinct reactions. Moreover, the number of enzymes and reactions catalyzed by members of the superfamily are increasing at an amazing pace as sequences of new genomes become available. The work described herein builds on and advances work from our laboratory on the characterization of some of the most novel reactions within the superfamily, including those involved in tRNA and ribosome modification, lipoic acid biosynthesis, the biosynthesis of several antibiotics, and antibiotic resistance. Specific objectives will be to i) elucidate how methylation of unactivated carbon and phosphorus atoms takes place, and provide rationale for the strategy employed for each type of methyl acceptor; ii) formulate methods to determine substrates for the many unannotated radical SAM methylases; iii) elucidate how iron-sulfur clusters are used as sources of sulfur atoms during sulfur insertion reactions and to determine how they are resynthesized after each turnover; iv) elucidate the pathway for the biosynthesis of the thiopeptide antibiotic, nosiheptide; and v) begin to characterize several radical SAM enzymes from humans that play important roles in health and disease.

项目概要 未活性碳中心的精制是酶催化的最苛刻的反应之一。 这些反应通常涉及自由基中间体，通常是通过底物的策略提取而产生的 氢原子(H·)。具有均裂键解离的 C-H 键断裂的普遍策略 能量 (BDE) 超过 95 kcal/mol 涉及由 O2 与过渡反应产生的中间体 金属辅助因子。一种独特的策略，在厌氧世界中占主导地位，在需氧世界中仍然很重要， 使用5'-脱氧腺苷5'-基团作为H·抽象基。该自由基是通过均分解产生的 腺苷钴胺素 (AdoCbl) 或 S-腺苷甲硫氨酸 (SAM) 的还原裂解。那些酶 利用 SAM 催化自由基依赖性反应属于所谓的自由基 SAM (RS) 超家族， 其中包含近 114,000 个单独的序列，涵盖至少 65 个不同的反应。而且， 超家族成员催化的酶和反应的数量正在以惊人的速度增加 随着新基因组序列的出现，步伐会加快。本文描述的工作建立在工作的基础上并推进工作 来自我们实验室对超家族中一些最新颖反应的表征， 包括参与 tRNA 和核糖体修饰、硫辛酸生物合成、 多种抗生素，以及抗生素耐药性。具体目标是 i) 阐明如何甲基化 发生未活性碳和磷原子，并为所采用的策略提供了理论依据 每种类型的甲基受体； ii) 制定方法来确定许多未注释自由基的底物 SAM 甲基化酶； iii) 阐明如何在硫过程中使用铁-硫簇作为硫原子的来源 插入反应并确定每次更新后它们如何重新合成； iv) 阐明途径 用于硫肽类抗生素那西肽的生物合成； v) 开始表征几个激进的 SAM 来自人类的酶在健康和疾病中发挥着重要作用。