Mechanism of a new flavin-derived cofactor involved in enzymatic decarboxylation reactions

通过这一奖项，化学部的生命过程化学计划资助密歇根大学的尼尔·马什博士研究一种潜在有用的新酶。酶是一种蛋白质，可以加速细胞内的许多化学反应，因此，酶是生命所必需的。由于酶具有高效、可再生、可生物降解和无毒等优点，人们对利用酶催化工业化学反应越来越感兴趣。脱羧酶是一类很有工业应用前景的酶。这些酶具有向分子中添加二氧化碳(或从分子中移除二氧化碳)的能力，这可能使它们能够用于开发新的生物燃料或重要的商品化学品，如苯乙烯(用于制造塑料)。本项目正在研究一种新发现的脱羧酶的生化性质，长期目标是生产这种用于工业应用的酶。与科学目标相配合，该项目正在推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。学生和博士后正在接受各种各样的专业技术培训，从有机合成到复杂的光谱学实验。该项目研究了最近从酵母中发现的一种新的两组分脱羧酶系统的机制。阿魏酸脱羧酶(FDC)催化多种取代苯基丙烯酸和山梨酸的脱羧基反应。该酶使用一种新的、修饰的黄素单核苷酸(FMN)作为辅因子，该辅因子包含一个额外的来自二甲基烯丙基磷酸酯的6元环。第二种酶，称为PAD1，从还原的FMN和二甲基烯丙基磷酸酯合成辅因子。特别是，Marsh小组的目的是确定FDC的机制和新型的、修饰的黄素辅因子在催化脱羧基反应中的作用。他们进一步的目的是确定PAD1合成修饰的黄素辅因子的机制。实验方法包括使用各种底物和辅酶类似物，使用稳态和预稳态技术来测量FDC和PAD1催化的反应的动力学。测量了FDC的氘、14C和13C动力学同位素效应，并与计算研究相结合，用酶过渡态模型拟合了实验确定的动力学同位素效应。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neil Marsh from the University of Michigan for studies on a potentially useful new enzyme. Enzymes are proteins that speed-up many chemical reactions within cells and thus, enzymes are necessary for life. There is increasing interest in using enzymes to catalyze industrial chemical reactions because they are extremely efficient, renewable, biodegradable and non-toxic. One class of industrially promising enzymes is known as decarboxylases. These enzymes have the ability to add carbon dioxide to (or remove carbon dioxide from) molecules, which may allow them to be used in processes to develop new biofuels or important commodity chemicals such as styrene (used in making plastics). This project is studing the biochemical properties of a newly discovered decarboxylase enzyme with the long-term objective of producing this enzyme for industrial applications. In synergy with the scientific goals, the project is advancing the education, training and professional development of undergraduates, graduate students and postdoctoral scientists in the inter-disciplinary area of chemical biology. Students and postdocs are receiving training in a wide variety of specialized techniques ranging from organic synthesis to sophisticated spectroscopy experiments. The project investigates the mechanism of a novel 2-component decarboxylase system recently identified from yeast. Ferulic acid decarboxylase (FDC) catalyzes the decarboxylation of a wide range of substituted phenylacrylic acids and sorbic acid. The enzyme uses a novel, modified flavin mononucleotide (FMN) as a cofactor that contains an additional 6-membered ring derived from dimethylallyl phosphate. The second enzyme, termed PAD1, synthesizes the cofactor from reduced FMN and dimethylallyl phosphate. In particular, the Marsh group aims to determine the mechanism of FDC and role of the novel, modified flavin cofactor in catalyzing the decarboxylation reaction. They further aim to determine the mechanism by which PAD1 synthesizes the modified flavin cofactor. The experimental approaches includes measuring the kinetics of the FDC- and PAD1-catalyzed reactions using both steady state and pre-steady state techniques with a variety of substrate and coenzyme analogs. Deuterium, and 14C and 13C kinetic isotope effects are measured for FDC and combined with computational studies to fit the experimentally-determined kinetic isotope effects with models of the enzyme transition state.