The Chemistry of Dihydropyrimidine Dehydrogenase

二氢嘧啶脱氢酶的化学性质

• 批准号: 2203593
• 负责人: Graham Moran
• 金额: $ 56.7万
• 依托单位: Loyola University of Chicago
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203593&HistoricalAwards=false
• 关键词: Chemistry; Dihydropyrimidine; Dehydrogenase

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Graham Moran from Loyola University of Chicago is studying the chemistry of the enzyme dihydropyrimidine dehydrogenase (DPD). The movement of electrons and hydrogen ions in biological processes is fundamental to the chemistry of life. The mechanism of transfer of electrons in biological systems involves complex networks of electron carriers and hydrogen ion donors and remains poorly understood. DPD transfers two electrons from an electron donor to reduce a base, which initiates degradation in a class of bases that includes DNA and RNA. DPD has a complex set of cofactors that transmit the electrons one at a time over long distances, which is unlike any other similar enzyme. The study of this enzyme is expected to advance the understanding of how charge transfer is used to favor specific reactions. The graduate and undergraduate students engaged in this research will gain knowledge and experience in spectrophotometry, transient state kinetics, and X-ray crystallography. Students will also learn in a yearly free virtual kinetics workshop that will illustrate how kinetic ideas are employed to glean information about the properties of molecules.DPD catalyzes the rate limiting step in the degradation of pyrimidine bases but it is significantly different from other dehydrogenases that accomplish similar chemistry. Specifically, DPD contains two flavin cofactors, FAD and FMN, separated by ~56 angstroms and connected by a molecular wire of four iron sulfur centers. The research plan is designed to test the hypotheses that pyrimidine reduction by DPD is the instigating step in the catalysis of base degradation and that NADPH is oxidized to backfill the enzyme and re-establish its two-electron reduced active state; in other words, the oxidative half reaction precedes the reductive half reaction. The research involves the modification of specific charged residues clustered around the FAD and FMN cofactors to slow or stall the passage of electrons through these junctions and thus to reveal different parts of the catalytic cycle. Specifically, mutations that reduce the number of negative charged residues proximal to the FAD will be used to raise the reduction potential of FAD and slow electron transfer to FMN. Similarly, mutations that reduce the number of positively charged residues near the FMN will be used to decrease its capacity to act as the electron sink. The research is designed in a way that makes it possible to track the movement of electrons and protons using combinations of variants with isotopic substitutions for substrates and solvent.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系生命过程化学（CLP）项目的支持下，芝加哥洛约拉大学的 Graham Moran 教授正在研究二氢嘧啶脱氢酶（DPD）的化学。生物过程中电子和氢离子的运动是生命化学的基础。生物系统中电子转移的机制涉及电子载体和氢离子供体的复杂网络，但人们对此仍知之甚少。 DPD 从电子供体转移两个电子以还原碱基，从而引发包括 DNA 和 RNA 在内的一类碱基的降解。 DPD 具有一组复杂的辅助因子，可以一次长距离传输一个电子，这与任何其他类似的酶不同。对这种酶的研究有望促进人们对如何利用电荷转移来促进特定反应的理解。从事这项研究的研究生和本科生将获得分光光度法、瞬态动力学和X射线晶体学方面的知识和经验。学生还将在每年一次的免费虚拟动力学研讨会上学习，该研讨会将说明如何利用动力学思想来收集有关分子特性的信息。DPD 催化嘧啶碱基降解中的限速步骤，但它与完成类似化学反应的其他脱氢酶有显着不同。具体来说，DPD 包含两种黄素辅因子 FAD 和 FMN，它们之间相距约 56 埃，并通过四个铁硫中心的分子线连接。该研究计划旨在测试以下假设：DPD 引起的嘧啶还原是碱基降解催化的引发步骤，以及 NADPH 被氧化以回填酶并重新建立其双电子还原活性状态；换句话说，氧化半反应先于还原半反应。该研究涉及对聚集在 FAD 和 FMN 辅因子周围的特定带电残基进行修饰，以减缓或阻止电子通过这些连接点的通道，从而揭示催化循环的不同部分。具体来说，减少靠近 FAD 的带负电残基数量的突变将用于提高 FAD 的还原电位并减缓电子向 FMN 的转移。类似地，减少 FMN 附近带正电残基数量的突变将被用来降低其作为电子汇的能力。该研究的设计方式使得可以使用同位素替代底物和溶剂的变体组合来跟踪电子和质子的运动。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。