Structure/Function Relationships in Cysteine and Cysteamine Dioxygenases

项目摘要 L-半胱氨酸(Cysteine-Cysteine，Cys)是新蛋白质生物合成的重要组成部分，是一种 几种生物上重要的含硫分子的前体，如辅酶A、牛磺酸、 谷胱甘肽和无机硫酸盐。然而，生物体必须严格控制其浓度 外源性半胱氨酸，因为这种半必需氨基酸水平的升高可能是极其有害的。非-- 血红素铁酶半胱氨酸双加氧酶(CDO)通过催化 半胱氨酸的氧化合成半胱氨酸亚磺酸。CDO失灵和随之而来的半胱氨酸积累 与几种神经退行性疾病有关。 辅酶A合成过程中半胱氨酸的脱羧基生成半胱胺。构成要素 辅酶A的降解释放这种半胱胺部分，它可以通过以下方式转化为次牛磺酸 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 随后氧化成牛磺酸，一种在哺乳动物中扮演许多重要角色的氨基硫醇酸 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CDO和ADO催化了两种结构相似的硫醇化合物的氧化，但它们表现出非常 底物的交叉利用效率低下。通过对CDO和ADO的并行研究，我们提出了一种 最终确定每种酶所采用的底物选择性机制的机会，从而 半胱氨酸和半胱胺水平在体内如何独立调节。《公约》的总体目标 因此，这项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 底物的选择性、定位和活化在CDO和ADO的催化机理中。 本着这一目标，我们制定了以下具体目标： 1.阐明CDO催化机理中的结构/功能关系。 2.评估真核生物和非生物之间关键保守氨基酸残基差异的影响 原核生物CDO活性部位/底物相互作用的性质。 3.建立底物半胱胺和O2与ADO活性部位结合的顺序和方式 以及硫醇氧化的机理。 4.探讨了CDO和ADO类化合物的几何/电子结构和反应机理。 为了实现这些目标，我们将使用生化、光谱和计算的组合 用于研究天然酶的静止状态和底物(类似物)加合物的工具，选择 变异体，以及小分子功能CDO和ADO的模拟物。

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.