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中心的酶催化一系列对医学和环境具有重要意义的反应。这项建议中描述的研究集中在最大的一组非血红素铁酶，铁(11)和α-酮戊二酸(AKG)依赖的羟基酶。我们试图更好地定义羟基酶反应中间体，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作用的知识。具体目标包括：(1)使用该酶家族研究得最好的代表酶TauD的变体，光谱分析Fe(IV)-氧代中间体，检查其他催化物种，并确定突变的蛋白质结构。此外，研究TauD与抑制剂的相互作用，并定量研究该酶的自羟化化学，以检验关于酶侧链修饰反应作用的假设。(2)阐明大肠杆菌DNA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进行结构和光谱表征，并利用结构和诱变方法确定两个相关酶RdpA和SdpA具有相反的酶特异性的基础。(4)确定一种新发现的氧化黄嘌呤的Fe(11)/AKG依赖的羟基酶的结构、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分析。(6)探索精选的Fe(11)/AKG羟基酶家族成员在分子筛去甲基化中发挥作用的可能性。前三个目标继续在实验室进行正在进行的研究，而后三个目标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，包括许多与直接医学相关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.