Study of alpha-ketoglutarate-dependent dioxygenases

α-酮戊二酸依赖性双加氧酶的研究

• 批准号: 6472966
• 负责人: ROBERT P HAUSINGER
• 金额: $ 23.46万
• 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/6472966
• 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.

描述（由申请人提供）：含有单核非血红素的酶 铁位点催化多种反应，这些反应对 医学和环境。该提案描述了研究三项的计划 最大但可能最不为人所知的群体的代表 这些酶：a-酮戊二酸 (aKG) 依赖性双加氧酶超家族。 TauD 第一步催化牛磺酸和其他磺酸盐的羟基化 在他们的新陈代谢中。 TfdA 在分解代谢过程中进行类似的化学反应 除草剂2,4-二氯苯氧基乙酸(2,4-D)。植酰辅酶A 羟化酶是本实验室的一个新的研究方向，需要 C-3支链脂肪酸的利用；这种人类酶的缺陷导致 Refsum 病、点状根茎软骨发育不良和 ZeHweger 综合征。 具体目标包括：（1）表征TauD的酶机制和 TfdA 通过检查催化中间体的性质并分析 定点诱变的影响。 (2) 检查酪氨酰的生物发生 TauD 中发现自由基、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。 (3) 获得 TauD、TfdA 和 TauD 的高分辨率三维蛋白质结构 它们在不同状态下的变体。 (4)探索金属中心 使用重组人酶测定植物酰辅酶A羟化酶的特性 和/或a.更易于处理的微生物模型系统。特别感兴趣的是 研究测试该酶超家族的新机制。具体来说，我们 提出这些酶具有催化必需的酪氨酸残基 用于将 Fe(IV)=O 共振稳定为 Fe(III)-OH/酪氨酸自由基 州。