Enzymatic Mechanism of Oxalate Decarboxylase Revealed by Biophysical and Structural Studies

有了这个奖项，化学部门的生命过程化学项目资助了佛罗里达大学的亚历山大·安格霍费尔博士，研究草酸脱羧酶对草酸的降解作用，这种酶在土壤细菌和真菌中发现。体内过量的草酸会导致肾结石和其他对动物和人类健康不利的影响。草酸作为一种植物产品，大量存在于许多植物性食品中。保持草酸含量较低的一种方法是通过酶催化的化学反应来分解草酸。Angerhofer博士的研究重点是阐明草酸脱羧酶分解草酸的机制。更具体地说，Angerhofer博士将测试一个假设，即为了分解草酸，这种蛋白质在两个锰离子之间进行长距离（约2纳米）的电子转移，其中一个锰离子位于草酸化学转化的活性位点。本研究将结合蛋白质工程的先进生化方法与结构和光谱学方法，包括x射线晶体学、电子顺磁共振和光谱学。这项研究的结果将增强我们对涉及生物活性锰的反应的基本知识，并最终有助于制定减轻植物中草酸存在的策略。该项目通过让学生参与研究，帮助他们获得在21世纪生物经济中有用的科学和专业技能，从而支持佛罗里达州不断增长的、多样化的科学和技术劳动力的培训。本科学生的培训是通过佛罗里达大学的基于课程的本科研究经验和大学研究学者计划来支持的，该计划将有天赋的新生带到现代研究的前沿。该奖项支持Angerhofer博士的研究，重点研究细菌酶草酸脱羧酶催化草酸中动态惰性碳-碳键断裂的分子机制。在蛋白质亚基的N端和c端锰离子之间的远程电子转移起着重要的催化作用。x射线晶体学、分子动力学模拟、电化学和一系列先进的电子顺磁共振（EPR）技术，结合定点诱变技术，将应用于这个问题。研究锰离子在蛋白质和电子传递活性氨基酸链中的活性。遗传密码扩展方法将用于将非天然氨基酸引入酶的特定目标位点，在那里它们可以用来探测酶的四级结构中可能存在的远程电子转移途径。计划中的实验将获得有关底物和氧辅因子与酶结合的重要结构和动力学信息。将要测试的假设是，通过远程电子转移，双氧通过在n端活性位点Mn离子上产生+3氧化态来促进催化，这反过来又驱动了反应。提出的工作将阐明蛋白质如何调节和利用单核锰中心的氧化还原电位进行催化。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Alexander Angerhofer from the University of Florida to investigate the degradation of oxalic acid by an enzyme known as oxalate decarboxylase which is found in soil bacteria and fungi. Overloading the body with oxalic acid can lead to kidney stones and other adverse health effects in animals and humans. As a plant product oxalic acid is present in many plant-derived foods in appreciable quantities. A way to keep the amount of oxalic acid low is to decompose it by chemical reactions catalyzed by enzymes. The research of Dr. Angerhofer is focused on the elucidation of the mechanism by which the enzyme oxalate decarboxylase breaks down oxalic acid. More specifically, Dr. Angerhofer will test the hypothesis that to decompose oxalic acid, this protein transfers electrons over a long distance, about 2 nm, between two manganese ions, one of which is located at the active site for the chemical transformation of oxalic acid. This study will involve use of a combination of advanced biochemical methods of protein engineering with structural and spectroscopic methods, including X-ray crystallography, electron paramagnetic resonance, and optical spectroscopy. The results of this research will enhance our fundamental knowledge of reactions that involve bio-active manganese and will ultimately aid in devising strategies to mitigate the presence of oxalic acid in plants. The project supports the training of a growing and diverse science and technology workforce in the state of Florida by involving students in research that helps them acquire scientific and professional skills useful in the bio-economy of the 21st century. The training of undergraduate students is supported through a course-based undergraduate research experience at the University of Florida and through the University Research Scholars Program, which brings gifted freshmen students to the cutting edge of modern research.This award supports the research of Dr. Angerhofer focused on the study of the molecular mechanisms by which the bacterial enzyme oxalate decarboxylase catalyzes the cleavage of the kinetically inert carbon-carbon bond in oxalic acid. It has been proposed that long-range electron transfer between the manganese ions situated at the N- and the C-terminal ends of the proteins subunits plays an important catalytic role. X-ray crystallography, molecular dynamics simulations, electrochemistry, and an array of advanced electron paramagnetic resonance (EPR) technologies, in combination with site-directed mutagenesis will be applied to the problem. The activity of manganese ions in the protein and a chain of electron transfer-active amino acids will be studied. Genetic code expansion methods will be used to introduce unnatural amino acids into the enzyme at specific target sites where they can be used to probe a long-range electron transfer pathway that may exist in the quaternary structure of the enzyme. The planned experiments will yield important structural and kinetic information about substrate and oxygen co-factor binding to the enzyme. The hypothesis that will be tested is that via long-range electron transfer dioxygen promotes catalysis by generating the +3 oxidation state on the active-site Mn ion situated at the N-terminal end, which in turn drives the reaction. The proposed work will elucidate how proteins tune and utilize the redox potential of mono-nuclear Mn centers for catalysis.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.