Switching Between One and Two Electron Mechanisms in the Nitroreductase Superfamily

硝基还原酶超家族中的一个和两个电子机制之间的切换

• 批准号: 10090611
• 负责人: STEVEN E ROKITA
• 金额: $ 31.64万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10090611
• 关键词: Active Sites; Affect; Antibiotic Therapy; Biochemical; Biochemistry; Biological; Catalysis; Categories; Chemical Structure; Chemicals; Chemistry; Complex; Crystallization; Data; Databases; Diagnosis; Diagnostic; Drug Targeting; Drug resistance; Electron Transport; Electrons; Enzymes; Family; Flavin Mononucleotide; Flavins; Foundations; Genes; Genomics; Industrialization; Investments; Iodide Peroxidase; Isotopes; Kinetics; Knowledge; Link; Measures; Methods; Molecular Conformation; Names; Nature; Nitroreductases; Oxidation-Reduction; Parents; Predictive Value; Process; Property; Proteins; Protons; Publishing; Research; Resistance; Scheme; Site-Directed Mutagenesis; Solvents; Source; Specificity; Structure; Subcategory; Testing; Virulence; Viscosity; analog; bioinformatics tool; cancer therapy; cofactor; dehalogenation; deiodination; fascinate; infancy; insight; member; predictive test; protonation; reconstitution

PROJECT SUMMARY/ABSTRACT Bioinformatic tools can offer exquisite insight into the ever growing quantities of genomic information. Gene sequences alone are usually sufficient to predict their membership in large structural superfamilies as well as more specific subfamilies that may share related functions. However, the scope of sequence data has so exceeded the realm of biochemical characterization that many subfamilies lack any structural, chemical or biological description. Strategies for predicting function within such groups are still in their infancy and will require a substantial investment in biochemistry to develop the essential links between sequence, structure and function. The nitroreductase superfamily offers an enticing platform to establish such a link to the redox chemistry of the flavin mononucleotide associated with these enzymes. This endeavor will capitalize on the extensive characterization of a few representative nitroreductases that lend their name to this superfamily and the recent release of a sequence similarity network that introduces a sophisticated order to more than 24,000 unique sequences. The iodotyrosine deiodinase group of this superfamily will now be investigated for its ability to promote sequential single electron transfer and suppress hydride transfer as a counterpoint to the ability of nitroreductases to act in the reverse by suppressing single electron transfer and promoting hydride transfer. Experimental strategies will include isotope and viscosity effects on catalysis by steady-state and rapid kinetics. Concurrently, substrate and flavin analogues will be used to measure the relative efficiencies of proton and electron transfer during deiodination. Key residues involved in these processes will then be identified by site-directed mutagenesis. A predictive understanding of flavin's redox chemistry will be constructed from these results and prior knowledge derived from nitroreductases. The principles developed by this comparison will be refined by two approaches. One will use structural and mechanistic data to direct conversion of a native deiodinase into a nitroreductase and vice versa. The other will examine the redox properties of superfamily members that have not yet been tested but can be anticipated from the correlations developed by this project.

项目概要/摘要 生物信息工具可以提供对不断增长的基因组数量的敏锐洞察 信息。单独的基因序列通常足以预测它们在大群体中的成员资格 结构超家族以及可能共享相关功能的更具体的亚家族。 然而，序列数据的范围已经超出了生化表征的范围，以至于 许多亚科缺乏任何结构、化学或生物学描述。预测策略 这些团体的职能仍处于起步阶段，需要大量投资 生物化学研究序列、结构和功能之间的重要联系。这 硝基还原酶超家族提供了一个诱人的平台来建立与氧化还原化学的联系 与这些酶相关的黄素单核苷酸。这一努力将利用 一些代表性硝基还原酶的广泛表征，由此得名 超家族和最近发布的序列相似性网络引入了复杂的 订购超过 24,000 个独特序列。 现在将研究该超家族的碘酪氨酸脱碘酶组的能力 促进连续的单电子转移并抑制氢化物转移作为对比 硝基还原酶通过抑制单电子转移和促进相反作用的能力 氢化物转移。实验策略将包括同位素和粘度对催化的影响 稳态和快速动力学。同时，底物和黄素类似物将用于测量 脱碘过程中质子和电子转移的相对效率。涉及的关键残留物 然后将通过定点诱变来识别这些过程。预测性理解 黄素的氧化还原化学将根据这些结果和源自的先验知识构建 硝基还原酶。通过这种比较制定的原则将通过两种方法进行完善。 人们将使用结构和机械数据直接将天然脱碘酶转化为脱碘酶 硝基还原酶，反之亦然。另一个将检查超家族成员的氧化还原特性 尚未经过测试，但可以从该项目开发的相关性中进行预测。