STRUCTURAL STUDIES ON EUKARYOTIC MOCO CONTAINING ENZYMES

含有钼辅助因子的酶在全球硫、氮和碳循环中催化重要的反应，目前被分为四个不同的家族。亚硫酸盐氧化酶和黄嘌呤脱氢酶是这四个家族中的两个关键成员。亚硫酸盐氧化酶催化生理活性的亚硫酸盐氧化成硫酸盐，在解毒外源亚硫酸盐和二氧化硫方面也起着重要作用。亚硫酸盐氧化酶缺乏症是人类的一种严重疾病，大多会导致儿童早期死亡。最近，亚硫酸盐氧化酶编码基因的第一个突变被鉴定出来。黄嘌呤脱氢酶是分解代谢嘌呤代谢的重要酶。黄嘌呤脱氢酶缺乏症称为黄嘌呤尿症，会导致不溶性黄嘌呤的积累，这可能会导致人类的急性肾功能衰竭。此外，黄嘌呤脱氢酶转化为酶的氧化酶形式后，与再灌注损伤有关。这项提案中概述的实验包括生化、生物物理和分子生物学方法的组合，目的是更好地了解酶及其与人类疾病的关系。这项建议的具体目标分为五类，概述如下：(1)重组大鼠和人亚硫酸盐氧化酶的结构特征：将确定大鼠和人的酶的野生型结构。这些结构将为亚硫酸盐氧化酶缺乏症的结构特征和突变的解释提供参考框架。(2)亚硫酸盐氧化酶缺乏症：我们将对迄今发现的7个突变体进行结构特征分析，并将它们与野生型结构进行比较，以确定影响酶活性的结构变化。(3)亚硫酸盐氧化酶的催化机理：通过对酶的无底物形式和抑制剂形式的结构表征，鉴定参与催化的残基，表征不同氧化状态下的钼配位构型，进一步深入了解亚硫酸盐氧化酶的催化作用。(4)电子转移机制：将研究分子内和分子间的电子转移机制，并确定对这两种途径都至关重要的残基。我们还将对亚硫酸盐氧化酶与其生理电子受体细胞色素c之间的络合物进行结构表征。(5)牛黄嘌呤脱氢酶的晶体结构分析：通过确定结合抑制剂的结构和确定NAD+结合形式来确定活性部位。

Enzymes containing the molybdenum cofactor catalyze important reactions in the global sulfur, nitrogen and carbon cycles and are currently classified into four different families. Sulfite oxidase and xanthine dehydrogenase are key members of two of these four families. Sulfite oxidase catalyzes the physiologically vital oxidation of sulfite to sulfate and also plays an important role in detoxifying exogenously supplied sulfite and sulfur dioxide. Sulfite oxidase deficiency is a severe disease in humans, which mostly leads to death in early childhood. Recently, the first mutations in the gene encoding sulfite oxidase have been identified. Xanthine dehydrogenase is an important enzyme in catabolic purine metabolism. Xanthine dehydrogenase deficiency known as xanthinuria causes an accumulation of insoluble xanthine, which can lead to acute renal failure in humans. Furthermore, xanthine dehydrogenase after conversion into the oxidase form of the enzyme has been implicated in reperfusion injury. The experiments outlined in this proposal, which comprise a combination of biochemical, biophysical and molecular biology methods, are aimed at a better understanding of both enzymes and their involvement in human diseases. The specific aims of this proposal are divided into five categories as outlined below: (1) Structural characterization of recombinant rat and human sulfite oxidase: The wildtype structures of the rat and human enzymes will be determined. These structures will provide the reference frame for the structural characterization and interpretation of the mutations, which lead to sulfite oxidase deficiency. (2) Sulfite oxidase deficiency: We will structurally characterize the seven so far identified mutants and compare them with the wildtype structures in order to characterize the structural changes, which compromise enzyme activity. (3) Catalytic mechanism of sulfite oxidase: We will identify residues which are involved in catalysis, characterize the Mo coordination geometry at different oxidation states and gain further insight into catalysis by structural characterization of substrate free and inhibitor forms of the enzyme. (4) Electron transfer mechanism: Both, the intra- and intermolecular electron transfer mechanisms will be studied and residues, which are crucial for either pathway will be identified. We will also structurally characterize the complex between sulfite oxidase and its physiological electron acceptor cytochrome c. (5) Crystal structure analysis of bovine xanthine dehydrogenase: Determination of the overall structure as well as characterization of the active site by determination of structures with bound inhibitors and determination of the NAD+ bound form.