Study of alpha-ketoglutarate-dependent dioxygenases

说明(申请人提供)：含有单核非血红素的酶 铁的位置催化了一系列重要的反应 对医学和环境的影响。这份提案描述了研究三个项目的计划 最大但可能最不为人所知的集团的代表 这些酶：依赖于α-酮戊二酸(AKG)的双加氧酶超家族。 作为第一步，TauD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了类似的化学研究 除草剂2，4-二氯苯氧基乙酸(2，4-D)。植酰辅酶A 羟基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；这种人类酶铅的缺陷 至Refsum病、根性点状软骨发育不良和ZeHweger综合征。 具体研究目标包括：(1)研究牛磺酸脱氢酶和牛磺酸脱氢酶的作用机制。 TFDA通过检测催化中间体的性质和分析 定点突变的效应。(2)酪氨酰的生物发生研究 TauD中发现的自由基、羟基-色氨酸和组氨基-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 它们的变种处于不同的状态。(4)探索金属分配者 重组人酶用于植酰辅酶A羟基酶的性质研究 和/或更易处理的微生物模型系统。特别值得关注的是 研究测试这种酶超家族的新机制。具体来说，我们 认为这些酶具有催化必需的酪氨酸残基 用于共振稳定Fe(IV)=O为Fe(III)-OH/酪氨酸自由基 各州。

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.