The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Experiments

先进EPR实验研究草酸脱羧酶的催化机制

• 批准号: 0809725
• 负责人: Alexander Angerhofer
• 金额: $ 43.15万
• 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: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0809725&HistoricalAwards=false
• 关键词: Catalytic; Mechanism; Oxalate; Decarboxylase; Studied

This research award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports work by Professor Alexander Angerhofer at the University of Florida to study the molecular mechanism by which the bacterial enzyme oxalate decarboxylase (OxDC) catalyzes the cleavage of the carbon-carbon bond in oxalic acid. Its presence in gut bacteria helps prevent the formation of kidney stones. Advanced electron paramagnetic resonance (EPR) techniques will be used in this project to study the electronic structure and local coordination environment of its two Mn-ions in the protein subunit. A multi-frequency approach using high magnetic fields of up to 25 Tesla will be used to identify the active site. These experiments will yield important kinetic, thermodynamic, and structural information about substrate and inhibitor binding to the enzyme and 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. Freeze-quench experiments will be employed to investigate the pre-equilibrium kinetics of enzyme-substrate interaction and yield time-dependent snapshots of the catalytic process. While the investigation of OxDC is of interest in its own right and may lead to future treatments of various oxalate-related pathologies (hyperoxaluria), the enzyme will also serve as an example of how the protein can modulate and affect catalytic reaction mechanisms. Site-directed mutants have been generated that have decreased oxalate decarboxylase but increased oxalate oxidase activity. Work with these mutants will help to elucidate how the structure of the active site can guide the chemistry. This project is tightly integrated into the HHMI-funded Science for Life initiative at the University of Florida which serves its undergraduate population by bringing them into the research lab early on. At least five of the most promising undergraduate science majors are expected to be involved in this project early in their years of study. They will receive intensive mentoring by Dr. Angerhofer and his collaborators and be given the opportunity to perform cutting-edge research which will not only enrich their education but also provide for them a perspective for a future career in the natural sciences.

无机，生物无机和有机化学计划的这项研究奖支持佛罗里达大学的亚历山大安格霍费尔教授的工作，以研究细菌酶草酸脱羧酶（OxDC）催化草酸中碳-碳键裂解的分子机制。它存在于肠道细菌中有助于防止肾结石的形成。本项目将采用先进的电子顺磁共振技术（EPR）研究其两个Mn离子在蛋白质亚基中的电子结构和局部配位环境。使用高达25特斯拉的高磁场的多频率方法将用于识别活动部位。这些实验将产生重要的动力学，热力学和结构信息的底物和抑制剂结合的酶和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。冷冻淬灭实验将用于研究酶-底物相互作用的预平衡动力学，并产生催化过程的时间依赖性快照。虽然OxDC的研究本身就很有意义，并且可能导致未来治疗各种与尿酸盐相关的疾病（高尿酸血症），但这种酶也将作为蛋白质如何调节和影响催化反应机制的一个例子。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。该项目与HHMI资助的佛罗里达大学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。预计至少有五名最有前途的本科理科专业学生将在学习初期参与该项目。他们将接受Angerhofer博士及其合作者的密集指导，并有机会进行尖端研究，这不仅会丰富他们的教育，而且还为他们提供了在自然科学未来职业生涯的视角。