STRUCTURAL STUDIES ON EUKARYOTIC MOCO CONTAINING ENZYMES

含有钼辅因子的酶催化全球硫、氮和碳循环中的重要反应，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家族的关键成员。 亚硫酸盐氧化酶催化生理上重要的亚硫酸盐氧化为硫酸盐，并且在解毒外源提供的亚硫酸盐和二氧化硫中也起重要作用。 亚硫酸盐氧化酶缺乏症是人类的一种严重疾病，主要导致儿童早期死亡。 最近，编码亚硫酸盐氧化酶的基因中的第一个突变已经被鉴定。黄嘌呤脱氢酶是嘌呤分解代谢的重要酶。 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，从而导致人类急性肾衰竭。 此外，黄嘌呤脱氢酶在转化为氧化酶形式的酶后与再灌注损伤有关。本提案中概述的实验包括生物化学、生物物理学和分子生物学方法的结合，旨在更好地了解酶及其与人类疾病的关系。 本提案的具体目标分为以下五类：（1）重组大鼠和人亚硫酸盐氧化酶的结构表征：将确定大鼠和人酶的野生型结构。 这些结构将为结构表征和解释突变提供参考框架，这导致亚硫酸盐氧化酶缺乏。 (2)亚硫酸盐氧化酶缺乏症：我们将从结构上表征迄今为止鉴定的七种突变体，并将其与野生型结构进行比较，以表征结构变化，这会损害酶活性。 (3)亚硫酸盐氧化酶的催化机制：我们将确定参与催化的残基，表征不同氧化态的Mo配位几何结构，并通过酶的无底物和抑制剂形式的结构表征进一步了解催化。 (4)电子转移机制：这两个，内和分子间的电子转移机制将进行研究和残基，这是至关重要的任何途径将被确定。 我们也将在结构上表征亚硫酸盐氧化酶和其生理电子受体细胞色素c之间的复合物。 (5)牛黄嘌呤脱氢酶的晶体结构分析：通过测定结合抑制剂的结构和测定NAD+结合形式，确定总体结构以及活性部位的表征。

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.