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

在化学学部生命过程化学（CLP）项目的支持下，密歇根大学的e·尼尔·g·马什教授正在研究一类新发现的酶如何将二氧化碳添加和去除到非活性分子中，使其更具化学活性。这项研究解决了迫切需要开发新的、环保的路线，从可再生能源中制造燃料和化学原料。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流程。酶是具有生物相容性、可生物降解性和无毒性的催化剂。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生产商品化学品。在科学目标的协同作用下，该项目将促进化学生物学跨学科领域的本科生、研究生和博士后科学家的教育、培训和专业发展，包括那些来自缺乏服务的少数群体的科学家。该项目有两个科学目标。首先是确定新的黄素辅助因子prFMN如何使用吩嗪-1-羧酸脱羧酶作为模型酶促进芳香分子的羧化。实验方法包括：原位天然蛋白质谱法鉴定底物与prFMN（戊烯基化黄素单核苷酸）之间形成的不稳定共价中间体；UV/可见光停流光谱用于识别瞬态形成的中间体并确定其形成和分解速率；天然丰度13-C动力学同位素效应测量用于确定断键和成键步骤的顺序，并提供过渡态性质的信息。其次是调查prFMN超家族中prFMN依赖性脱羧反应的多样性，目的是鉴定新的底物和新的（脱）羧化酶。实验方法将包括使用含有芳香、杂环或共轭双键功能的化合物文库筛选潜在的羧基化底物。当与适当的酶孵育时，潜在的底物将通过它们进行H/D交换的能力来识别。许多化合物将使用LC-MS（液相色谱-质谱检测）并行筛选。该方法将应用于采样prFMN超家族的不同横截面。这些研究有望为这类重要酶的机制提供基础信息。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.