Structure function investigations of radical transfer and disulfide exchange in a class Ia ribonucleotide reductase

Ia类核糖核苷酸还原酶自由基转移和二硫键交换的结构功能研究

• 批准号: 10386378
• 负责人: Dana Westmoreland
• 金额: $ 6.72万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386378
• 关键词: Active Sites; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Antiviral Agents; Area; Biochemical; Biological; Biological Assay; C-terminal; Catalysis; Chemicals; Chemistry; Competence; Complex; Coupled; Crystallization; Crystallography; Cysteine; DNA Repair; DNA biosynthesis; Data; Deoxyribonucleotides; Development; Dissociation; Disulfides; Drug Targeting; Electron Microscopy; Electron Transport; Electrons; Enzymes; Escherichia coli; Generations; Institution; International; Investigation; Iron; Knowledge; Label; Location; Maps; Mentorship; Methods; Monitor; Mutation; NADP; Oxidation-Reduction; Pathogenicity; Pathway interactions; Peptides; Pharmaceutical Preparations; Process; Proteins; Protons; Publications; Publishing; Reaction; Research; Resolution; Ribonucleotide Reductase; Ribonucleotide Reductase Subunit; Ribonucleotides; Role; Science; Side; Site; Site-Directed Mutagenesis; Spectrum Analysis; Structure; TXN gene; Tail; Techniques; Time; Training; Tryptophan; Tyrosine; Variant; Viral; Water; Work; analog; anti-cancer; antineoplastic antibiotics; base; cofactor; collaborative environment; cryogenics; novel; skills; structural biology; thioredoxin reductase; water channel

PROJECT SUMMARY/ABSTRACT Class Ia ribonucleotide reductases (RNRs) are anti-cancer and antibiotic drug targets due to their role in the biosynthesis of deoxyribonucleotides. They convert ribonucleotides to deoxyribonucleotides using radical- based chemistry with reducing equivalents coming from a pair of cysteine residues in the active site that form a disulfide upon enzyme turnover. Recent structural work elucidating the active state structure of the class Ia RNR of Escherichia coli has revealed for the first time, the intact 32-Å-long pathway for radical transfer between subunits that is responsible for the generation of a catalytically essential radical species in the enzyme active site. In this proposed research, I will investigate the role of water channels in the proton transfer that occurs concomitant with electron transfer. I will also investigate the role of a tryptophan residue (W48) in a radical transfer pathway that otherwise involves tyrosine residues. Furthermore, I will probe the structural rearrangements that occur in order to facilitate re-reduction of the active site disulfide. This re-reduction pathway is known to be dependent on the C-terminal tail of the α subunit of RNR, which shuttles reducing equivalents from thioredoxin to the active site, but no structural snapshots of this process have ever been captured. Here, I propose to use site-specific mutagenesis to investigate the involvement of water channels in proton transfer by assessing the catalytic competency of RNR variants in which the water networks have been disrupted. I intend to assess the role of W48 in the PCET pathway through the site-specific incorporation of unnatural W analogues which will give us a spectroscopic handle with which to directly monitor radical transfer to and from W48. Finally, I propose to capture structural snapshots of the re-reduction steps of RNR, including a structure of RNR with thioredoxin using cryogenic electron microscopy. These studies will enable us to answer several of the major outstanding questions of this prototypical RNR. This work will also enable me, a physical inorganic and materials chemist by training, to gain expertise in biochemical and structural biology methods which will inform new research directions that will benefit from a synthesis of these two skill sets. As one of the world’s foremost research institutions, MIT is an ideal and highly collaborative environment in which to pursue the studies described in this proposal. Furthermore, as an internationally recognized leader in the field of structural biology, Prof. Drennan’s scientific mentorship will enable me to excel in my undertaking of this research.

项目摘要/摘要 IA类核糖核苷酸还原酶（RNR）是抗癌和抗生素药物靶标，因为它们在 脱氧核糖核苷酸的生物合成。他们使用自由基 - 从一对半胱氨酸保留在形成A的活性部位的基于化学的基础化学反应 酶离职后的二硫化物。最近阐明了IA类的主动状态结构的结构性工作 大肠杆菌的RNR首次揭示了完整的32-Å长途径 在负责产生催化性自由基物种的亚基之间 酶活性位点。在这项拟议的研究中，我将研究水通道在质子转移中的作用 这与电子传递同时发生。我还将调查色氨酸居住（W48）在 根本转移途径，否则涉及酪氨酸残留物。此外，我将探究结构 为了促进重新减少主动部位二硫化物的重新排列。这种重新减少 已知途径取决于RNR的α亚基的C末端尾巴，该途径可穿梭还原 从硫氧还蛋白到活跃部位的等效物，但没有该过程的结构快照 被捕获。在这里，我建议使用特定位点特异性诱变来研究水通道参与 质子转移通过评估水网络的RNR变体的催化能力 破坏了。我打算通过特定地点的工作来评估W48在PCET途径中的作用 不自然的W类似物将为我们提供一个直接监视自由基转移的光谱手柄 往返W48。最后，我建议捕获RNR的重新还原步骤的结构快照，包括 使用低温电子显微镜与硫氧还蛋白的RNR结构。这些研究将使我们能够 回答该原型RNR的几个主要问题。这项工作也将使我 通过培训进行物理无机和材料化学家，以获取生化和结构生物学方面的专业知识 将为新的研究方向提供信息的方法，这些方法将受益于这两种技能集的综合。作为 麻省理工学院是世界上最重要的研究机构之一，是一个理想且高度协作的环境 追求本提案中描述的研究。此外，作为国际认可的领导人 结构生物学领域，Drennan教授的科学心态将使我能够在这方面表现出色 研究。