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.

在该化学过程的化学过程奖中，化学分部的化学过程计划中，佛罗里达大学的亚历山大·愤怒博士将研究分子机制，细菌酶Oxalate脱羧酶（OXDC）和特定地点指导的突变体催化了氧化碳酸碳键的裂解。该酶在控制草酸的控制中可能具有重要的应用，这是最常见的天然毒素和肾结石中主要成分之一。晚期电子顺磁共振（EPR）和电子核双共振（ENDOR）技术将用于研究发生催化的蛋白质亚基中两个MN离子的电子结构和局部协调环境。旋转诱捕将用于研究酶促转移期间从蛋白质中释放的自由基。将使用高场EPR研究底物和各种抑制剂与酶的结合。这些实验将产生有关底物和抑制剂与酶结合的重要动力学，热力学和结构信息，并将检验当前的工作假设，其中MN和相关结合的氧分子在Oxalate C-C键破裂中充当了氧化还原穿梭。还将研究蛋白质中远程电子转移链的可能存在及其在催化过程中的作用。虽然OXDC本身就是感兴趣的，并且可能导致对各种草酸盐相关病理（高黄油，肾结石等）的未来治疗，但该酶也将作为蛋白质如何控制反应性自由基中间体的一个例子。已经产生了降低草酸脱羧酶但草酸盐氧化酶活性的位置突变体。与这些突变体一起工作将有助于阐明活性位点的结构如何指导化学和脱羧酶和氧化酶活性之间的切换。该项目紧密整合到佛罗里达大学的霍华德·休斯医学研究所“生命科学”倡议中，该计划通过早期将其带入研究实验室来为其本科生群体提供服务。每年平均两个最有前途的本科科学专业的专业将在大二或大三的夏季直接支持该项目。他们将有额外的志愿者机会，并向学位进行学分的研究，这使他们能够在高三期间写荣誉论文。这些学生将获得Angerhofer博士及其合作者的密集指导，并有机会进行尖端的研究，这不仅会丰富他们的教育，而且还为他们提供了自然科学未来职业的观点。他们还将有机会在地方，地区和民族科学会议上介绍他们的研究。 Angerhofer博士将尽一切努力从STEM学科中人数不足的团体中招募学生。