Spectroscopic, Structural and Kinetic Characterization of Sulfite Oxidase from Mu

Mu 亚硫酸盐氧化酶的光谱、结构和动力学表征

• 批准号: 7333722
• 负责人: Kayunta Johnson-Winters
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333722
• 关键词: Amino Acids; Animals; Anions; Attenuated; Biochemical; Brain; Cells; Cessation of life; Charge; Chickens; Crystalline Lens; Crystallization; Cysteine; Data; Defect; Electron Spin Resonance Spectroscopy; Electron Transport; Enzymes; Escherichia coli; Frequencies; Goals; Growth; Heme; House mice; Human; Inherited; Inorganic Sulfates; Joint Dislocation; Kinetics; Laboratories; Lasers; Length; Libraries; Link; Membrane Proteins; Mental Retardation; Metabolism; Methionine; Modification; Molybdenum; Mouse-ear Cress; Mus; Mutation; Nervous System Trauma; Nuclear; Oxidases; Physiologic pulse; Plants; Point Mutation; Property; Pulse taking; Recombinants; Research; Resolution; Roentgen Rays; Sequence Homology; Series; Source; Structure; Sulfites; Sulfur; Symptoms; System; Thinking; Unspecified or Sulfate Ion Sulfates; Variant; analog; flash photolysis; inhibitor/antagonist; insight; molybdenum cofactor; mutant; oxidation; protein expression; research study; tool

DESCRIPTION (provided by applicant): Sulfite Oxidase (SO) is a molybdenum cofactor dependent enzyme that catalyzes the oxidation of sulfite to sulfate as the final step in the degradation of the sulfur-containing amino acids, cysteine and methionine. SO is physiologically vital in human metabolism, and hereditary sulfite oxidase deficiency results in severe neurological damage with symptoms that include dislocation of the ocular lenses, attenuated growth of the brain, mental retardation and early death. SO deficiency results either from a defect in the synthesis of the molybdenum cofactor or from various point mutations with the enzyme. While SO's from several sources have been previously studied, there is no comprehensive set of structural, spectroscopic and reactivity data from a common source. The primary goal of the research presented in this proposal is to gain greater insight into the function and mechanism of SO and the effect of point mutations through an integrated series of biochemical and biophysical studies of SO from Mus musculus (house mouse), which is readily expressed in DL41 plus E. coli cells. Mouse SO is 82% identical with the human enzyme, but has not been previously investigated extensively. The primary tools for investigating the properties of mutants (variants) of mouse SO will be: 1) Laser flash photolysis studies of intramolecular electron transfer (IET) between the molybdenum domain and the b type heme domain; 2) high resolution variable frequency pulsed EPR experiments to determine the structure of the transient catalytic molybdenum center through nuclear couplings (1H, 2H, 31P, 17O and 33S); 3) crystallization and X-ray structure determination as a function of pH and anions in the media. Despite numerous attempts in other laboratories, intact human SO has proven difficult to crystallize. To date the only crystal structure for intact animal SO is the 1997 structure of wild-type chicken SO. For human SO, the only available structural information is for the isolated heme domain. Plant SO, from Arabidopsis thaliana only has a molybdenum domain. Thus, there is a need for an animal system that has a high sequence homology to human SO and for which substantial quantities of enzyme variants can be systematically produced for study. Mouse SO meets these criteria and preliminary data on expression of the protein show the feasibility of the proposed studies.

描述（由申请方提供）：亚硫酸盐氧化酶（SO）是一种钼辅因子依赖性酶，可催化亚硫酸盐氧化为硫酸盐，这是含硫氨基酸、半胱氨酸和蛋氨酸降解的最后一步。SO在人体代谢中具有重要的生理学意义，遗传性亚硫酸氧化酶缺乏症会导致严重的神经损伤，症状包括晶状体脱位、大脑发育迟缓、智力迟钝和早死。SO缺乏症是由于钼辅因子的合成缺陷或酶的各种点突变引起的。虽然以前已经研究了来自几个来源的SO，但是没有来自共同来源的结构、光谱和反应性数据的综合集合。本研究的主要目的是通过对小鼠SO的一系列生物化学和生物物理学研究，更深入地了解SO的功能和机制以及点突变的影响。coli细胞。小鼠SO与人类酶有82%的相同性，但以前没有进行过广泛的研究。研究突变体特性的主要工具小鼠SO（变种）将：1）激光闪光光解研究钼结构域和B型血红素结构域之间的分子内电子转移（IET）; 2）高分辨率变频脉冲EPR实验，通过核偶联确定瞬时催化钼中心的结构（1H，2 H，31 P，17 O和33 S）; 3）结晶和X射线结构测定作为介质中pH和阴离子的函数。尽管在其他实验室进行了许多尝试，但完整的人类SO已被证明难以结晶。迄今为止，完整的动物SO的唯一晶体结构是1997年野生型鸡SO的结构。对于人类SO，唯一可用的结构信息是孤立的血红素域。来自拟南芥的植物SO仅具有钼结构域。因此，需要与人SO具有高序列同源性的动物系统，并且可以系统地产生大量的酶变体用于研究。小鼠SO符合这些标准，蛋白质表达的初步数据显示了拟议研究的可行性。