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

在化学系生命过程化学项目的支持下，E。尼尔·G来自密歇根大学的马什正在研究一种新发现的酶如何将二氧化碳添加到不反应的分子中，使其更具化学反应性。 这项研究解决了开发新的环境友好路线的迫切需要，以从可再生资源中制造燃料和化学原料。 了解这些酶如何工作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。酶是生物相容的、可生物降解的和无毒的催化剂。在工业过程中使用酶和生物可再生原料有助于以成本效益高、能耗低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。 该项目将配合科学目标，在化学生物学跨学科领域促进本科生、研究生和博士后科学家，包括来自得不到充分服务的少数群体的科学家的教育、培训和专业发展。 第一个是确定新的黄素辅因子，prFMN，促进（脱）使用吩嗪-1-羧酸脱羧酶作为模型酶的芳香分子的羧化。 实验方法包括：原位天然蛋白质质谱鉴定底物和prFMN之间形成的不稳定共价中间体（异戊二烯化黄素单甘肽）; UV/可见光停流光谱法，用于识别瞬时形成的中间体并确定其形成和分解速率，以及天然丰度13-C动力学同位素效应测量，用于确定键断裂和键形成步骤的顺序，并提供关于过渡状态的性质的信息。 第二个是调查的多样性prFMN依赖的脱羧反应中所代表的prFMN超家族，识别新的底物和新的（脱）羧酶的目的。 实验方法将涉及使用含有芳香族、杂环或共轭双键官能团的化合物库筛选用于羧化的潜在底物。 通过与适当酶孵育时进行H/D交换的能力来鉴别潜在底物。 将使用LC-MS（液相色谱-质谱检测）平行筛选许多化合物。 该方法将被应用于采样的prFMN超家族的不同的横截面。 这些研究有望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.