Structure/Function Relationships in Cysteine and Cysteamine Dioxygenases

半胱氨酸和半胱胺双加氧酶的结构/功能关系

• 批准号: 9751323
• 负责人: Thomas Christian Brunold
• 金额: $ 27.14万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751323
• 关键词: Acids; Active Sites; Address; Alzheimer' s Disease; Amino Acids; Anabolism; Arginine; Autoimmune Diseases; Binding; Biochemical; Biological; Brain; Catalysis; Coenzyme A; Collaborations; Comparative Study; Complex; Crystallization; Crystallography; Cysteamine; Cysteine; Cysteine dioxygenase; Cystine; Cystinuria; Data; Decarboxylation; Development; Dioxygenases; Disease; Disulfides; Duodenum; Effectiveness; Electrons; Environment; Enzyme Kinetics; Enzymes; Excretory function; Exhibits; Face; Generations; Glutamine; Glutathione; Glycine; Goals; Growth; Human; Hydrogen Peroxide; Inorganic Chemistry; Inorganic Sulfates; Ischemia; Kidney; Lead; Ligands; Link; Mammals; Membrane; Methodology; Mind; Modeling; Molecular; Molecular Biology; Motor Neuron Disease; Nature; Neurodegenerative Disorders; Neurons; Neurotransmitters; Organism; Oxides; Parkinson Disease; Pathway interactions; Play; Positioning Attribute; Process; Production; Proteins; Rattus; Reaction; Research; Rest; Role; Skeletal Muscle; Specificity; Structure; Structure-Activity Relationship; Substrate Interaction; Substrate Specificity; Sulfhydryl Compounds; Sulfur; Taurine; Teratogens; Testing; Tissues; Triad Acrylic Resin; Tyrosine; Variant; adduct; analog; carboxylate; computerized tools; cross reactivity; crosslink; cysteine sulfinic acid; developmental disease; electronic structure; enzyme mechanism; excitotoxicity; heart function; hypotaurine; in vivo; insight; nervous system disorder; oxidation; scaffold; semi essential amino acid; small molecule; three dimensional structure

Project Summary L-Cysteine (Cys) is an essential building block for the biosynthesis of new proteins and serves as a precursor for several biologically important sulfur-containing molecules, such as coenzyme A, taurine, glutathione, and inorganic sulfate. However, organisms must tightly regulate the concentration of exogenous Cys, as elevated levels of this semi-essential amino acid can be extremely harmful. The non- heme iron enzyme cysteine dioxygenase (CDO) serves to maintain the proper Cys levels by catalyzing the oxidation of Cys to cysteine sulfinic acid. Malfunctioning of CDO and the consequent accumulation of Cys have been linked to several neurodegenerative diseases. The decarboxylation of Cys during coenzyme A synthesis generates cysteamine. The constitutive degradation of coenzyme A releases this cysteamine moiety, which can be converted to hypotaurine by the non-heme iron enzyme cysteamine (2-aminoethanethiol) dioxygenase (ADO). Hypotaurine is subsequently oxidized to taurine, an amino thiol acid that plays numerous important roles in mammalian tissues, including maintaining cardiac functions, protecting neural cells from excitotoxicity and ischemia, serving as a neurotransmitter, and stabilizing skeletal muscle membrane. Despite catalyzing the oxidation of two structurally similar thiol compounds, CDO and ADO show very inefficient cross-utilization of substrates. By studying CDO and ADO in parallel, we are presented with an opportunity to conclusively determine the substrate selectivity mechanism each enzyme employs, and thus how Cys and cysteamine levels may be independently regulated in vivo. The overall objective of the research outlined in this proposal is, therefore, to identify the roles of key amino acid residues with regards to substrate selectivity, positioning, and activation in the CDO and ADO catalytic mechanisms. With this objective in mind, we have devised the following Specific Aims: 1. Elucidate structure/function relationships in the catalytic mechanism of CDO. 2. Assess the effects of differences in key conserved amino acid residues between eukaryotic and prokaryotic CDOs on the nature of active site/substrate interactions. 3. Establish the order and modes by which the substrates cysteamine and O2 bind to the ADO active site and the mechanism of thiol oxidation. 4. Explore the geometric/electronic structures and reaction mechanisms of CDO and ADO mimics. To accomplish these aims, we will employ a combination of biochemical, spectroscopic, and computational tools for studying the resting states and substrate (analogue) adducts of the native enzymes, select variants, and small-molecule functional CDO and ADO mimics.

项目概要 L-半胱氨酸 (Cys) 是新蛋白质生物合成的重要组成部分，可作为 几种生物学上重要的含硫分子的前体，例如辅酶 A、牛磺酸、 谷胱甘肽和无机硫酸盐。然而，生物体必须严格调节 外源性半胱氨酸，因为这种半必需氨基酸水平升高可能极其有害。非 血红素铁酶半胱氨酸双加氧酶 (CDO) 通过催化 Cys 氧化成半胱氨酸亚磺酸。 CDO 故障以及随之而来的 Cys 积累 与多种神经退行性疾病有关。 辅酶 A 合成过程中 Cys 脱羧生成半胱胺。本构 辅酶 A 的降解释放半胱胺部分，可通过以下方式转化为亚牛磺酸： 非血红素铁酶半胱胺（2-氨基乙硫醇）双加氧酶（ADO）。亚牛磺酸是 随后氧化为牛磺酸，这是一种氨基硫醇酸，在哺乳动物中发挥着许多重要作用 组织，包括维持心脏功能，保护神经细胞免受兴奋性毒性和缺血， 作为神经递质，稳定骨骼肌膜。 尽管催化两种结构相似的硫醇化合物的氧化，CDO 和 ADO 表现出非常好的催化作用。 基材交叉利用效率低下。通过并行研究 CDO 和 ADO，我们得到了 有机会最终确定每种酶采用的底物选择性机制，从而 如何在体内独立调节半胱氨酸和半胱胺水平。总体目标 因此，本提案中概述的研究是确定关键氨基酸残基在以下方面的作用： CDO 和 ADO 催化机制中的底物选择性、定位和活化。 考虑到这一目标，我们制定了以下具体目标： 1. 阐明CDO催化机制中的结构/功能关系。 2. 评估真核生物和真核生物之间关键保守氨基酸残基差异的影响 原核 CDO 对活性位点/底物相互作用性质的影响。 3. 建立底物半胱胺和O2与ADO活性位点结合的顺序和模式 以及硫醇氧化的机理。 4. 探索CDO和ADO模拟物的几何/电子结构和反应机制。 为了实现这些目标，我们将结合使用生化、光谱和计算技术 用于研究天然酶的静息状态和底物（类似物）加合物的工具，选择 变体，以及小分子功能性 CDO 和 ADO 模拟物。