STRUCTURAL STUDIES ON EUKARYOTIC MOCO CONTAINING ENZYMES

含钼辅助因子的酶在全球硫、氮和碳循环中催化重要反应，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两个的关键成员。亚硫酸盐氧化酶催化亚硫酸盐的生理活性氧化，并在解毒外源亚硫酸盐和二氧化硫中起重要作用。亚硫酸盐氧化酶缺乏症是一种严重的人类疾病，主要导致儿童早期死亡。最近，亚硫酸盐氧化酶编码基因的第一个突变已经被确定。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，即黄嘌呤尿，会引起不溶性黄嘌呤的积累，从而导致人类急性肾功能衰竭。此外，转化为氧化酶形式的黄嘌呤脱氢酶与再灌注损伤有关。本提案中概述的实验包括生物化学、生物物理和分子生物学方法的结合，目的是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：将确定大鼠和人亚硫酸盐氧化酶的野生型结构。这些结构将为结构表征和解释导致亚硫酸盐氧化酶缺乏症的突变提供参考框架。(2)亚硫酸盐氧化酶缺乏症：我们将对迄今鉴定的7个突变体进行结构表征，并将其与野生型结构进行比较，以表征影响酶活性的结构变化。(3)亚硫酸盐氧化酶的催化机理：我们将确定参与催化的残基，表征不同氧化态下的Mo配位几何形状，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。我们还将从结构上表征亚硫酸盐氧化酶与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：通过确定结合抑制剂的结构和确定NAD+的结合形式来确定总体结构和活性位点的表征。

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.