Enzymatic Mechanism of Oxalate Decarboxylase Revealed by Biophysical and Structural Studies

生物物理和结构研究揭示草酸脱羧酶的酶机制

• 批准号: 2002950
• 负责人: Alexander Angerhofer
• 金额: $ 47.3万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2002950&HistoricalAwards=false
• 关键词: Enzymatic; Mechanism; Oxalate; Decarboxylase; Revealed

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.

该奖项将资助佛罗里达大学的Alexander Angerhofer博士研究一种名为草酸脱羧酶的酶对草酸的降解作用，这种酶存在于土壤细菌和真菌中。体内草酸过量会导致肾结石和其他对动物和人类健康的不利影响。草酸作为一种植物产品，大量存在于许多植物性食品中。保持低草酸含量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，安格霍费尔博士将测试一个假设，即为了分解草酸，这种蛋白质会在两个锰离子之间传递电子，距离很远，约为2 nm，其中一个位于草酸化学转化的活性部位。这项研究将结合使用先进的蛋白质工程生化方法与结构和光谱方法，包括X射线结晶学、电子顺磁共振和光学光谱学。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础知识，并最终将有助于制定策略，以减少植物中草酸的存在。该项目通过让学生参与研究，帮助他们获得对21世纪生物经济有用的科学和专业技能，从而支持佛罗里达州不断增长和多样化的科学和技术劳动力的培训。本科生的培训通过佛罗里达大学以课程为基础的本科生研究经验和大学研究学者计划得到支持，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，他专注于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。有人认为，位于蛋白质亚基N-端和C-端的锰离子之间的长程电子转移起着重要的催化作用。X射线结晶学、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，以及定点突变将被应用于这个问题。将研究蛋白质和一系列具有电子转移活性的氨基酸中锰离子的活性。遗传密码扩展方法将被用来在特定的靶点将非天然氨基酸引入酶中，在那里它们可以用来探测可能存在于酶的四级结构中的远程电子转移途径。计划中的实验将产生关于底物和氧辅助因子与酶结合的重要结构和动力学信息。将被检验的假设是，通过远程电子转移，氧气通过在位于N-末端的活性中心锰离子上产生+3氧化态来促进催化，进而推动反应。这项拟议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潜力作为催化剂。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

选择性掺入 5-羟色氨酸可阻断草酸脱羧酶中的长程电子转移

• DOI: 10.1002/pro.4537
• 发表时间: 2022
• 期刊: Protein Science
• 影响因子: 8
• 作者: Pastore, Anthony John;Montoya, Alvaro;Kamat, Manasi;Basso, Kari B.;Italia, James S.;Chatterjee, Abhishek;Drosou, Maria;Pantazis, Dimitrios A.;Angerhofer, Alexander
• 通讯作者: Angerhofer, Alexander

Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.

• DOI: 10.1021/acs.biochem.1c00164
• 发表时间: 2021-10-26
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Li Q;Zallot R;MacTavish BS;Montoya A;Payan DJ;Hu Y;Gerlt JA;Angerhofer A;de Crécy-Lagard V;Bruner SD
• 通讯作者: Bruner SD