Mechanism and Scope of Prenylated-flavin-dependent (De)carboxylase Enzymes

在化学系生命过程化学(CLP)计划的支持下，密歇根大学的E.Neil G.Marsh教授正在研究一种新发现的酶如何在非活性分子中添加和移除二氧化碳，以使它们更具化学活性。这项研究解决了开发新的、环境友好的路线的迫切需要，以利用可再生资源制造燃料和化学品原料。了解这些酶的工作原理将有助于开发高效的“绿色”催化剂，以取代目前的能源密集型工业过程。酶是生物相容、可生物降解、无毒的催化剂。在工业过程中使用酶和生物可再生原料有助于以具有成本效益、低能耗和可持续的方式取代化石碳氢化合物用于生产商品化学品。与科学目标相配合，该项目将促进化学生物学跨学科领域的本科生和研究生以及博士后科学家的教育、培训和专业发展，其中包括那些来自服务不足的少数群体的科学家。第一个是以吩嗪-1-羧酸脱羧酶为模型酶，确定新型黄素辅因子prFMN如何促进芳香族分子的(去)羧化。实验方法包括：原位天然蛋白质质谱仪，以确定底物和prFMN(PrFMN)之间形成的不稳定的共价中间体；UV/可见光停流光谱，以确定瞬时形成的中间体并确定其形成和破裂速率；以及自然丰度13-C动力学同位素效应测量，以确定键断裂和键形成步骤的顺序，并提供关于过渡态的性质的信息。第二个是考察prFMN超家族中依赖于prFMN的脱羧基反应的多样性，目的是确定新的底物和新的(去)羧基酶。实验方法将包括使用包含芳香族、杂环或共轭双键官能团的化合物文库筛选潜在的羧化底物。潜在的底物将根据它们在与适当的酶孵育时进行H/D交换的能力来确定。许多化合物将使用LC-MS(液-质联用检测)进行并行筛选。该方法将被应用于PrFMN超家族的不同横截面样本。这些研究预计将提供关于这类重要酶的机制的基本信息。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Chemistry Division, Professor E. Neil G. Marsh from the University of Michigan is studying how a newly-discovered class of enzymes add and remove carbon dioxide to unreactive molecules to make them more chemically reactive. This research addresses an urgent need to develop new, environmentally friendly routes to make fuels and chemical feedstocks from renewable sources. Understanding how these enzymes work will aid in developing highly efficient “green” catalysts to replace current energy-intensive industrial processes. Enzymes are biocompatible, biodegradable and non-toxic catalysts. Using enzymes and biorenwable feedstocks in industrial processes helps to replace fossil hydrocarbons for the production of commodity chemicals in ways that are cost-effective, low-energy and sustainable. In synergy with the scientific goals, the project will advance the education, training and professional development of undergraduate and graduate students, and postdoctoral scientists, including those from underserved minority groups, in the interdisciplinary area of chemical biology.The project has two scientific goals. The first is to determine how the novel flavin cofactor, prFMN, facilitates (de)carboxylation of aromatic molecules using phenazine-1-carboxylate decarboxylase as a model enzyme. The experimental approaches include: in situ native protein mass spectrometry to identify unstable covalent intermediates formed between substrates and prFMN (prenylated flavin mononucleotide); UV/visible stopped-flow spectroscopy to identify transiently formed intermediates and determine their rates of formation and breakdown and natural abundance 13-C kinetic isotope effect measurements to determine the order of bond-breaking and bond-forming steps, and to provide information on the nature of the transition state. The second is to survey the diversity of prFMN-dependent decarboxylation reactions represented in the prFMN superfamily, with the objective of identifying novel substrates and new (de)carboxylases. The experimental approach will involve screening potential substrates for carboxylation using compound libraries that contain aromatic, heterocyclic, or conjugated double bond functionalities. Potential substrates will be identified by their ability to undergo H/D exchange when incubated with the appropriate enzyme. Many compounds will be screened in parallel using LC-MS (liquid chromatography with mass spectral detection). The approach will be applied to sample a diverse cross-section of the prFMN superfamily. These studies are expected to provide fundamental information about the mechanisms of this important class of enzymes.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.