Study of alpha-ketoglutarate-dependent dioxygenases

α-酮戊二酸依赖性双加氧酶的研究

• 批准号: 6731125
• 负责人: ROBERT P HAUSINGER
• 金额: $ 25.42万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731125
• 关键词: 5 hydroxytryptophan; Raman spectrometry; catalyst; circular dichroism; dihydroxyphenylalanine; electron nuclear double resonance spectroscopy; enzyme biosynthesis; enzyme mechanism; enzyme structure; high performance liquid chromatography; iron; metalloenzyme; oxoglutarate dehydrogenase; oxygenases; protein binding; site directed mutagenesis; stop flow technique; tyrosine; ultraviolet spectrometry

DESCRIPTION (provided by applicant): Enzymes containing mononuclear non-heme iron sites catalyze a diverse array of reactions that are significant to medicine and to the environment. This proposal describes plans to study three representatives of the largest, but perhaps least well understood, grouping of these enzymes: The a-ketoglutarate (aKG)-dependent dioxygenase superfamily. TauD catalyzes the hydroxylation of taunne and other sulfonates as a first step in their metabolism. TfdA carries out similar chemistry during catabolism of the herbicide 2,4-dichiorophenoxyacetic acid (2,4-D). Phytanoyl-CoA hydroxylase, a new research direction for this laboratory, is required for utilization of C-3 branched fatty acids; deficiencies of this human enzyme lead to Refsum disease, rhizomelic chondrodysplasia punctata, and ZeHweger syndrome. The specific aims include: (1) Characterize the enzyme mechanism of TauD and TfdA by examining the properties of catalytic intermediates and analyzing the effects of site-directed mutagenesis. (2) Examine the biogenesis of the tyrosyl radical, hydroxy-tryptophan, and histidyl-trihydroxyphenylalanine found in TauD and identify the structures & synthesis of modifications present in TfdA. (3) Obtain high-resolution three-dimensional protein structures of TauD, TfdA, and their variants in their various states. (4) Explore the metallocenter properties of phytanoyl-CoA hydroxylase using the recombinant human enzyme and/or a. more tractable microbial model system. Of particular interest will be studies to test a new mechanism for this enzyme superfamily. Specifically, we propose that these enzymes possess catalytically essential tyrosine residues that are used to resonance stabilize Fe(IV)=O as Fe(III)-OH/tyrosine radical states.

性状（由申请方提供）：含单核非血红素的酶 铁位点催化各种各样的反应， 医学和环境。该提案描述了研究三个 最大的，但也许是最不了解的， 这些酶：α-酮戊二酸（aKG）依赖性双加氧酶超家族。 TauD催化牛磺酸和其他磺酸盐的羟基化作为第一步 在他们的新陈代谢中。TfdA在催化过程中进行类似的化学反应， 除草剂2，4-二氯苯氧乙酸（2，4-D）。植烷酰辅酶A 羟化酶是本实验室新的研究方向， C-3支链脂肪酸的利用;这种人类酶的缺乏导致 Refsum病、点状肢根软骨发育不良和ZeHweger综合征。 具体目标包括：（1）研究TauD的酶促作用机制， TfdA通过检查催化中间体的性质和分析 定点诱变的效果。(2)检查酪氨酰的生物发生 在TauD中发现的自由基、羟基色氨酸和组氨酰三羟基苯丙氨酸 并鉴定TfdA中存在的修饰的结构和合成。（三） 获得TauD、TfdA和TfdB的高分辨率三维蛋白质结构。 他们在不同国家的变体。(4)探索购物中心 使用重组人酶的植烷酰辅酶A羟化酶的性质 和/或a.更易处理的微生物模型系统。特别令人感兴趣的是 研究测试这种酶超家族的新机制。我们特别 提出这些酶具有催化必需的酪氨酸残基 用于共振稳定Fe（IV）=O作为Fe（III）-OH/酪氨酸自由基 states.