The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Techniques

先进EPR技术研究草酸脱羧酶催化机制

• 批准号: 1213440
• 负责人: Alexander Angerhofer
• 金额: $ 43万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1213440&HistoricalAwards=false
• 关键词: Catalytic; Mechanism; Oxalate; Decarboxylase; Studied

In this award from the Chemistry of Life Processes Program in the Chemistry Division, Dr. Alexander Angerhofer, from the University of Florida, will study the molecular mechanisms by which the bacterial enzyme oxalate decarboxylase (OxDC) and specific site-directed mutants catalyze the cleavage of the carbon-carbon bond in oxalic acid. This enzyme may have important applications in the control of oxalic acid which is one of the most common naturally occurring toxins and the main ingredient in kidney stones. Advanced electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) techniques will be used to study the electronic structure and local coordination environment of two Mn ions in the protein subunit where catalysis takes place. Spin trapping will be used to investigate free radicals that are released from the protein during enzymatic turnover. Binding of substrate and various inhibitors to the enzyme will be studied with high-field EPR. These experiments will yield important kinetic, thermodynamic, and structural information about binding of substrate and inhibitors to the enzyme, and will test the current working hypothesis in which Mn and an associated bound oxygen molecule act as redox shuttles in the breaking of the oxalate C-C bond. The possible existence of a long-range electron transfer chain in the protein and its role in the catalytic process will also be investigated. While OxDC is of interest in its own right and may lead to future treatments of various oxalate-related pathologies (hyperoxaluria, kidney stones, etc.), the enzyme will also serve as an example of how a protein can control reactive radical intermediates. Site-directed mutants have been generated that have decreased oxalate decarboxylase but increased oxalate oxidase activites. Work with these mutants will help to elucidate how the structure of the active site can guide the chemistry and switch between decarboxylase and oxidase activities. This project is tightly integrated into Howard Hughes Medical Institute "Science for Life" initiative at the University of Florida, which serves its undergraduate student population by bringing them into research labs early on. Each year on average two of the most promising undergraduate science majors will be directly supported in this project in the summer of their sophomore or junior years. They will have additional volunteer opportunities and perform research for credit toward their degrees which allows them to write an honors thesis during their senior year. These students will receive intensive mentoring by Dr. Angerhofer and his collaborators and will be given the opportunity to perform cutting-edge research, which will not only enrich their education but also provide them a perspective for a future career in the natural sciences. They will also have the opportunity to present their research at local, regional, and national scientific conferences. Dr. Angerhofer will make every effort to recruit students from groups that are underrepresented in the STEM disciplines.

在化学部生命过程计划的化学奖中，来自佛罗里达大学的亚历山大安格霍费尔博士将研究细菌酶草酸脱羧酶（OxDC）和特定定点突变体催化草酸中碳-碳键裂解的分子机制。这种酶可能在草酸的控制中具有重要的应用，草酸是最常见的天然毒素之一，也是肾结石的主要成分。先进的电子顺磁共振（EPR）和电子核双共振（ENDOR）技术将被用来研究的电子结构和本地的两个锰离子在蛋白质亚基的催化发生的配位环境。自旋捕获将用于研究酶促周转期间从蛋白质释放的自由基。将用高场EPR研究底物和各种抑制剂与酶的结合。这些实验将产生重要的动力学，热力学和结构信息结合的底物和抑制剂的酶，并将测试目前的工作假设，其中锰和相关的结合氧分子作为氧化还原穿梭在草酸C-C键的断裂。还将研究蛋白质中可能存在的长程电子转移链及其在催化过程中的作用。虽然OxDC本身是令人感兴趣的，并且可能导致未来治疗各种与尿酸盐相关的病理（高尿酸血症、肾结石等），该酶也将作为蛋白质如何控制活性自由基中间体的实例。已经产生了定点突变体，其具有降低的草酸脱羧酶但增加的草酸氧化酶活性。对这些突变体的研究将有助于阐明活性位点的结构如何指导化学反应以及脱羧酶和氧化酶活性之间的转换。该项目与佛罗里达大学的霍华德休斯医学研究所的“生命科学”计划紧密结合，该计划通过将本科生早期带入研究实验室来服务本科生群体。平均每年两名最有前途的本科理科专业学生将在大二或大三的夏天直接获得该项目的支持。他们将有额外的志愿者机会，并对他们的学位进行研究，使他们能够在大四期间撰写荣誉论文。Angerhofer和他的合作者，并将有机会进行前沿研究，这不仅会丰富他们的教育，也为他们提供了一个在自然科学的未来职业生涯的角度。他们还将有机会在地方，区域和国家科学会议上展示他们的研究。Angerhofer将尽一切努力从STEM学科代表性不足的群体中招募学生。