Exploring the Mechanistic Diversity of Prenylated-Flavin-Dependent Enzymes

有必要开发新的、环境友好的途径，从可再生资源中获得燃料和化学原料。有了这个奖项，化学部的生命过程化学计划正在资助密歇根大学安阿伯的Neil Marsh博士研究新发现的一类酶如何从不反应的起始材料中去除二氧化碳，使其更具化学反应性，并可能形成增值的商品化学品。了解这些酶的基本反应机理将有助于开发高效的"绿色"催化剂，从而有可能取代能源密集型工业过程。这种生物相容、生物可降解和无毒的催化剂与替代化石碳氢化合物的努力相吻合，以生产具有成本效益、低能耗和可持续的商品化学品。 在科学目标的协同作用下，该项目推进了本科生和研究生的教育、培训和专业发展，和博士后科学家该项目将阐明三种异戊烯基化黄素（prFMN）的作用机制，脱羧酶似乎使用相同的辅因子通过三种不同的机制催化脱羧反应。 这些酶是：阿魏酸脱羧酶，其中假设prFMN通过偶极环加成机制反应; 4-羟基苯甲酸脱羧酶，其中prFMN可以通过亲电子机制反应;以及吩嗪-1-羧酸脱羧酶，其中证据表明prFMN可以通过自由基机制反应。实验方法包括稳态和预稳态动力学分析结合同位素标记的基板。 这些研究确定了酶中间体的形成和分解速率。 这些研究还使用新的质谱方法鉴定了底物和prFMN之间形成的不稳定共价中间体，这些方法允许分析天然状态下的蛋白质。 最后，研究人员将探索与底物类似物的反应途径，这些底物类似物可能作为基于机制的抑制剂发挥作用。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。

There is a need to develop new, environmentally friendly routes to fuels and chemical feedstocks from renewable sources. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neil Marsh from the University of Michigan Ann Arbor to examine how a newly-discovered class of enzymes remove carbon dioxide from unreactive starting materials to make them more chemically reactive and likely to form value-added commodity chemicals. Understanding the basic mechanisms of reaction for these enzymes will allow the development of highly efficient "green" catalysts to potentially replace energy-intensive industrial processes. Such biocompatible, biodegradable and non-toxic catalysts dovetail with efforts to replace fossil hydrocarbons for the production of commodity chemicals that are cost-effective, low-energy and sustainable. In synergy with the scientific goals, the project advances the education, training and professional development of undergraduate and graduate students, and postdoctoral scientists (including those from underrepresented minority groups) in the interdisciplinary area of chemical biology.The project will elucidate the mechanisms of three prenylated-flavin (prFMN) decarboxylases that appear to use the same cofactor to catalyze decarboxylation reactions through three distinct mechanisms. These enzymes are: ferulic acid decarboxylase in which prFMN is hypothesized to react by a dipolar cycloaddition mechanism; 4-hydroxybenzoate decarboxylase in which prFMN may react by an electrophilic mechanism; and phenazine-1-carboxylate decarboxylase for which evidence suggests prFMN may react by a radical mechanism. The experimental approaches include steady state and pre-steady state kinetic analyses combined with isotopically-labelled substrates. These studies determine the rates of formation and breakdown of enzyme intermediates. The researches also identify unstable covalent intermediates formed between substrates and prFMN using novel mass spectrometry methods that allow for the analysis of proteins in their native state. Finally, the researchers will probe the reaction pathways with substrate analogs that may function as mechanism-based inhibitors.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.