Characterization fo Fe(II)alpha-ketoglutarate-dependent hydroxylases

Fe(II)α-酮戊二酸依赖性羟化酶的表征

• 批准号: 7869489
• 负责人: ROBERT P HAUSINGER
• 金额: $ 10.5万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7869489
• 关键词: Alkanesulfonates; Biochemical; Chemicals; Chemistry; Chromatin; Computer-Assisted Image Analysis; DNA; Environment; Enzymes; Escherichia coli; Family; Family member; Heme Iron; Herbicides; Homologous Gene; Human; Hydroxylation; Investigation; Knowledge; Laboratories; Medical; Medicine; Mixed Function Oxygenases; Modification; Mononuclear; Mutagenesis; Property; Proteins; Reaction; Research; Research Personnel; Role; Side; Site; Structure; Testing; Variant; Xanthines; alpha ketoglutarate; base; demethylation; inhibitor/antagonist; insight; mutant; programs; protein structure; repair enzyme; taurine-alpha-ketoglutarate dioxygenase

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. The studies described in this proposal focus on the largest group of non-heme iron enzymes, the Fe(ll)- and alpha-ketoglutarate (aKG)-dependent hydroxylases. We seek to better define the hydroxylase reaction intermediates, enhance our understanding of the protein features involved in substrate recognition, and expand our knowledge of the functional roles of related family members. The specific aims include: (1) Use variants of the best-studied representative of this enzyme family, the sulfonate-metabolizing enzyme TauD, to spectroscopically analyze the Fe(IV)-oxo intermediate, examine other catalytic species, and to determine mutant protein structures. Also, investigate TauD interactions with inhibitors and study quantitatively the self-hydroxylation chemistry of this enzyme to test hypotheses regarding the role of the enzyme side chain modification reactions. (2) Elucidate the interactions of the E. coli DMA-repair enzyme AlkB with its substrate, methylated DNA, and investigate the roles of several human homologues. (3) Structurally and spectroscopically characterize the herbicide-degrading enzyme TfdA, and determine the basis for the opposite enahtiospecificities of two related enzymes, RdpA and SdpA, by using structural and mutagenesis approaches. (4) Define the structure, biochemical properties, and spectroscopically accessible catalytic intermediates of a newly identified Fe(ll)/aKG-dependent hydroxylase that oxidizes xanthine. (5) Identify the function of the E. coli Gab protein and spectroscopically examine its catalytic intermediates. (6) Explore the potential for carefully selected Fe(ll)/aKG hydroxylase family members to function in criromatin demethylation. The first three aims continue ongoing investigations in the laboratory, while the latter three aims present new research directions. Insights gained from these studies will be useful in understanding the substrate recognition features and chemical mechanisms of a large number of enzymes, including many less tractable examples that have direct medical relevance.

描述（由申请人提供）：含有单核非血红素位点的酶催化​​对医学和环境重要的各种反应。该提案中描述的研究重点是最大的非血红素铁酶，Fe（LL）和α-酮戊二酸（AKG）依赖性羟基酶。我们试图更好地定义羟化酶反应中间体，增强我们对涉及底物识别的蛋白质特征的理解，并扩展我们对相关家庭成员功能作用的了解。具体目的包括：（1）使用该酶家族最佳代表的变体，磺酸盐代谢酶TAUD，以光谱分析Fe（IV） - 氧中间体，检查其他催化物种，并确定突变蛋白结构。此外，研究与抑制剂的相互作用，并定量研究该酶的自羟基化化学，以检验有关酶侧链修饰反应作用的假设。 （2）阐明了大肠杆菌DMA-REPAIR酶ALKB与其底物甲基化DNA的相互作用，并研究了几种人类同源物的作用。 （3）在结构和光谱上，通过使用结构和诱变方法来确定两种相关酶RDPA和SDPA的相反enahtiospixies的基础，并确定相反的Enahtiospifies的基础。 （4）定义新鉴定的Fe（LL）/Akg依赖性羟化酶的结构，生化特性和光谱可访问的催化中间体，可氧化Xanthine。 （5）确定大肠杆菌GAB蛋白的功能，并通过光谱检查其催化中间体。 （6）探索精心选择的Fe（LL）/Akg羟化酶家族的潜力，使其在criromatin脱甲基化中起作用。前三个目标继续在实验室进行的调查，而后三个目标则提出了新的研究指示。从这些研究中获得的见解将有助于理解大量酶的底物识别特征和化学机制，包括许多具有直接医学相关性的较低术的例子。