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Biochemical Studies of Oxalate Decarboxylase

草酸脱羧酶的生化研究

基本信息

批准号：
7624484
负责人：
Nigel Gordon RICHARDS
金额：
$ 20.49万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7624484
关键词：
ATP Synthesis Pathway Active Sites Aerobic Anaerobic Bacteria Binding Biochemical Biochemistry Bioinorganic Chemistry Blood Carbon Dioxide Catalysis Chemicals Chimera organism Classification Clinical Treatment Complex Computing Methodologies Condition Decarboxylation Development Dioxygen Disease Electron Spin Resonance Spectroscopy Environment Enzyme Kinetics Enzymes Evolution Exhibits Formates Frequencies Goals Human Ions Isotopes Kinetics Knowledge Ligands Literature Location Manganese Measurement Mediating Metals Methods Modeling Molecular Molecular Evolution Numbers Object Attachment Oxalate decarboxylase Oxalates Oxalic Acid Oxalic Acids Oxalobacter formigenes Oxalyl-CoA decarboxylase Oxidases Oxidation-Reduction Play Prevention Process Property Protein Engineering Proteins Reaction Regulation Research Roentgen Rays Role Series Side Site-Directed Mutagenesis Spectrum Analysis Structure Techniques Testing Therapeutic Translational Research Tryptophan Urine Variant Work X-Ray Crystallography arginylglutamate computational chemistry computer studies density enzyme activity formyl-coenzyme A transferase insight microorganism mutant novel oxalate oxidase oxidation protein folding research study small molecule theories

项目摘要

Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human pathological conditions associated with the accumulation of this compound in the blood and/or urine. This proposal outlines the continuation of integrated experimental and computational studies aimed at understanding the fundamental biochemistry and regulation of oxalate decarboxylase (OxDC), an enzyme that catalyzes the conversion of oxalate to carbon dioxide and formate. The goals of this project are (i) to test mechanistic proposals for the involvement of radical species in the molecular processes that result in cleavage of the oxalate C-C bond, (ii) to investigate the role of dioxygen and higher oxidation states of the active site manganese ion in the catalytic mechanism, and (iii) to evaluate changes in local protein environment function so as to modulate the intrinsic reactivity of the Mn(II) center in the enzyme. An integrated experimental and computational strategy, using techniques in bioinorganic chemistry, molecular spectroscopy, enzyme kinetics and protein engineering, X-ray crystallography, and computational chemistry, will be pursued in these efforts, which have two major specific aims. In the first aim, biophysical structural methods, EPR spectroscopy and density functional theory (DFT) calculations will be used to validate the hypothetical mechanism of OxDC-catalyzed decarboxylation, and establish the precise manganese oxidation state that mediates the reaction. The second aim will focus on the kinetic and biophysical characterization of OxDC mutants in an effort to evaluate the effect of protein environment in modulating the chemical reactivity of the Mn(II) center(s) in the enzyme. In addition to the general biochemical implications of these efforts for our knowledge of the chemical mechanisms by which enzymes can catalyze difficult reactions, and the molecular processes that seem to be used in evolving proteins with altered catalytic properties, these efforts should provide a platform for long-term translational research on the development of novel therapies for the clinical treatment and/or prevention of oxalate-related disease.

可以催化草酸分解的酶在治疗中具有潜在的应用。治疗与该化合物在血液中积累有关的人类病理状况和/或尿。该提案概述了综合实验和计算研究的继续旨在了解草酸脱羧酶（OxDC）的基本生物化学和调节，催化草酸盐转化为二氧化碳和甲酸盐的酶。该项目的目标是 (i)测试自由基物种参与分子过程的机制建议，导致草酸酯C-C键的裂解，（ii）研究分子氧和更高氧化态的作用，活性位点锰离子在催化机制，和（iii）以评估局部蛋白质的变化环境功能，以便调节酶中Mn（II）中心的固有反应性。集成实验和计算策略，使用生物无机化学，分子光谱学，酶动力学和蛋白质工程，X射线晶体学和计算化学，将继续进行在这些努力中，有两个主要的具体目标。在第一个目标，生物物理结构方法，EPR 光谱和密度泛函理论（DFT）计算将用于验证假设 OxDC催化脱羧的机理，并建立精确的锰氧化态，调节反应。第二个目标将集中在动力学和生物物理特性的OxDC 突变体，以评估蛋白质环境在调节蛋白质的化学反应性中的作用。 Mn（II）中心。除了这些努力的一般生物化学影响外，了解酶可以催化困难反应的化学机制，以及酶的分子结构。这些过程似乎用于进化具有改变的催化性质的蛋白质，这些努力应该为临床开发新疗法的长期转化研究提供平台治疗和/或预防与糖尿病相关的疾病。