The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Techniques

来自佛罗里达大学的Alexander Angerhofer博士将研究细菌酶草酸脱羧酶（OxDC）和特定位点定向突变体催化草酸中碳-碳键断裂的分子机制。这种酶可能在控制草酸方面有重要的应用，草酸是最常见的自然毒素之一，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）技术将用于研究发生催化作用的蛋白质亚基中两个Mn离子的电子结构和局部配位环境。自旋捕获将用于研究在酶周转过程中从蛋白质中释放的自由基。底物和各种抑制剂与酶的结合将用高场EPR进行研究。这些实验将获得关于底物和抑制剂与酶结合的重要动力学、热力学和结构信息，并将测试目前的工作假设，即Mn和相关的结合氧分子在草酸C-C键的断裂中充当氧化还原穿梭体。还将研究蛋白质中可能存在的远程电子传递链及其在催化过程中的作用。虽然OxDC本身就很有趣，并且可能导致未来各种草酸盐相关疾病（高草酸尿、肾结石等）的治疗，但该酶也将作为蛋白质如何控制活性自由基中间体的一个例子。位点导向突变体已经产生，草酸脱羧酶降低，但草酸氧化酶活性增加。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脱羧酶和氧化酶活性之间的化学反应和转换。该项目与佛罗里达大学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，为他们提供服务。每年平均有两名最有前途的理科生将在他们大二或大三的夏天直接得到这个项目的支持。他们将有额外的志愿者机会，并进行研究以获得学位学分，这使他们能够在大四时写一篇荣誉论文。这些学生将接受Angerhofer博士及其合作者的密集指导，并将有机会进行前沿研究，这不仅将丰富他们的教育，还将为他们未来在自然科学领域的职业生涯提供一个视角。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成果。Angerhofer博士将尽一切努力从STEM学科中代表性不足的群体中招募学生。

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.