Exploring the Mechanistic Diversity of Prenylated-Flavin-Dependent Enzymes

有必要开发新的、环境友好的路线，从可再生资源中获取燃料和化学品原料。该奖项由密歇根大学安娜堡分校的生命过程化学项目资助来自密歇根大学安娜堡分校的Neil Marsh博士研究一种新发现的酶如何从不起作用的原料中去除二氧化碳，使它们具有更强的化学活性，并有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，以潜在地取代能源密集型工业过程。这种生物相容、可生物降解和无毒的催化剂与取代化石碳氢化合物生产具有成本效益、低能耗和可持续的商品化学品的努力相吻合。与科学目标相协调，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(包括那些来自未被充分代表的少数群体的科学家)的教育、培训和专业发展。该项目将阐明三种异丙基黄素脱羧酶(PrFMN)的机制，这三种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化反应。这些酶是：阿魏酸脱羧酶，其中prFMN被假设为通过偶极环加成反应；4-羟基苯甲酸脱羧酶，其中prFMN可能通过亲电机理反应；以及吩嗪-1-羧酸脱羧酶，证据表明prFMN可能通过自由基机制反应。实验方法包括结合同位素标记底物的稳态和稳态前动力学分析。这些研究确定了酶中间体的形成和分解速度。这些研究还使用新的质谱学方法确定了底物和prFMN之间形成的不稳定的共价中间体，这些方法允许分析蛋白质的天然状态。最后，研究人员将探索底物类似物的反应路径，这些底物类似物可能起到基于机制的抑制剂的作用。这一奖项反映了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。

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.