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.

在化学过程（CLP）方案的支持下，芝加哥洛约拉大学的格雷厄姆·莫兰（Graham Moran）教授正在研究二氢吡咪定脱氢酶（DPD）的化学。电子和氢离子在生物过程中的运动至关重要。电子在生物系统中转移的机制涉及电子载体和氢离子供体的复杂网络，并且仍然知之甚少。 DPD从电子供体传输两个电子以减少碱，从而在包括DNA和RNA的一类碱基中降解。 DPD具有一组复杂的辅助因子，该辅因子一次在长距离时一次传输电子一个，这与其他类似的酶不同。对该酶的研究有望提高人们对电荷转移如何使用特定反应的理解。从事这项研究的研究生和本科生将获得分光光度法，瞬态状态动力学和X射线晶体学方面的知识和经验。学生还将在年度免费的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催化了嘧啶基碱基降解的速率限制步骤，但与其他脱氢酶的降解差异很大。具体而言，DPD包含两个黄素辅因子，即FAD和FMN，被〜56埃，并由四个铁硫中心的分子线连接。该研究计划旨在检验假设，即DPD减少嘧啶是基础降解催化的煽动步骤，并且NADPH被氧化以回填酶并重新建立其两电子的降低活性状态。换句话说，氧化的半反应先于还原的半反应。该研究涉及修改围绕FAD和FMN辅助因子聚集的特定电荷残基，以减慢或失速电子通过这些连接，从而揭示催化循环的不同部分。具体而言，减少近端为FAD的负电荷残基数量的突变将用于提高FAD和缓慢的电子转移到FMN的降低潜力。同样，减少FMN附近带正电荷残基数量的突变将用于降低其作为电子水槽的能力。这项研究的设计方式使得使用与同位素替代的底物和溶剂的组合跟踪电子和质子的运动。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛影响的审查标准来通过评估来通过评估来支持的。