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

描述（由申请人提供）：含有单核非血红素铁位点的酶催化一系列对医学和环境有重要意义的反应。本提案中描述的研究集中在最大的非血红素铁酶组，铁(ll)-和α -酮戊二酸（aKG）依赖性羟化酶。我们试图更好地定义羟化酶反应中间体，增强我们对底物识别所涉及的蛋白质特征的理解，并扩大我们对相关家族成员功能角色的认识。具体目标包括：(1)使用该酶家族中研究得最好的代表，即磺酸代谢酶TauD的变体，对Fe(IV)-oxo中间体进行光谱分析，检查其他催化物种，并确定突变蛋白结构。此外，研究TauD与抑制剂的相互作用，并定量研究该酶的自羟基化化学，以测试有关酶侧链修饰反应作用的假设。(2)阐明大肠杆菌dma -修复酶AlkB与其底物甲基化DNA的相互作用，并研究几种人类同源物的作用。(3)对除草剂降解酶TfdA进行了结构和光谱表征，并通过结构和诱变方法确定了两种相关酶RdpA和SdpA的相反特异性的基础。(4)确定新发现的Fe(ll)/ akg依赖性羟化酶氧化黄嘌呤的结构、生化性质和光谱可及的催化中间体。(5)鉴定大肠杆菌Gab蛋白的功能，并对其催化中间体进行光谱检测。(6)探索精心挑选的Fe(ll)/aKG羟化酶家族成员在犯罪染色质去甲基化中的作用。前三个目标在实验室中继续进行研究，而后三个目标则提出了新的研究方向。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不太容易处理的例子。

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.