半胱氨酸双加氧酶是一类非卟啉环的单核铁金属酶，它催化半胱氨酸转化为半胱氨酸亚磺酸的氧化反应。在该反应过程中，来自于同一个底物双氧分子的两个氧原子被加在了底物半胱氨酸的巯基硫上。这个不可逆氧化反应是一系列重要代谢通路的起始，牵涉到丙酮酸、硫酸、亚牛磺酸和牛磺酸等重要的小分子化合物。半胱氨酸双加氧酶在哺乳动物的脑、肾、肺等组织中均有可检出的表达水平，在肝脏中有高水平表达。在肝脏细胞中，半胱氨酸双加氧酶对于保持细胞内适当的游离半胱氨酸水平起着重要的作用。临床研究表明，过高的游离半胱氨酸水平与多种神经退行性疾病和自免疫疾病相关。自从1966年以来，哺乳动物半胱氨酸双加氧酶的功能已经被研究得相当透彻了，但其催化机制仍不清楚。虽然近年来先后有小鼠、大鼠和一种细菌的半胱氨酸双加氧酶结构被解析得到，并据此提出了一些催化机制，但由于没有在结构中观察到底物的结合，因而这些机制缺乏直接实验证据的支持。 本论文首次报导了人源半胱氨酸双加氧酶与底物半胱氨酸的复合物晶体结构。基于对该结构的分析，构建了人半胱氨酸双加氧酶的七个单点突变体，并对其进行了酶学和金属含量方面的研究。在酶学研究方面，强调了氧气作为底物的重要地位，改进了酶活测定的研究体系。所有这些研究结果为半胱氨酸双加氧反应机制的研究提供了深入的视角。半胱氨酸双加氧酶的催化机制与一个罕见的硫醚键紧密相关。这个硫醚键是由来自于蛋白质的一个酪氨酸残基和一个半胱氨酸残基通过共价键而形成的。同样的硫醚键此前只在半乳糖氧化酶的结构中被报导过。在半胱氨酸双加氧酶中，该硫醚键扮演了完全不同的角色，包括：对亚铁离子的稳定；协助氧－氧单键的断开；防止高危害性羟自由基的出现。半胱氨酸双加氧酶催化机制的提出，为针对该酶的病理学和药物学研究奠定了基础。
Cysteine dioxygenase (CDO, EC 1.13.11.20) is a non-heme mononuclear iron metalloenzyme which catalyzes the oxidation of cysteine to cysteine sulfinic acid with addition of molecular dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolism pathways related to sulfurate compounds, including pyruvate, sulfate, hypotaurine and taurine. CDO is expressed at appreciable levels in the brain, kidney, and lung with extremely higher levels in the liver tissue, where CDO plays an important role in maintaining the hepatic concentration of intracellular free cysteine within a proper narrow range. Clinical evidence suggests that elevated cysteine levels are involved in several nerve-degeneration diseases and autoimmunity diseases. The function of mammalian CDOs has been studied in stark since 1966. Its mechanism, nonetheless, remains unknown. Recently, several crystal structures of murine, rat and necator CDO were reported and resulted in some proposals on its catalytic mechanism. Unfortunately, no substrate small molecules are found in these previously reported structures, leading to a lack of direct evidence for those proposals. We solved the first crystal structure of human CDO in complex with its substrate cysteine. Based on preliminary analysis of this structure, several single-amino-acid mutants of human CDO were constructed to perform both enzymatic activity and metal content assays. We emphasized the important role of substrate dioxygen molecules, and improved the methods for enzymatic assays of CDO. All the results provide an insight into the mechanism of cysteine thiol oxidation catalyzed by CDO, which is tightly associated with a thioether-bonded tyrosine-cysteine cofactor. Before the structure of CDO was reported, same Tyr-Cys cofactor was only found in the crystal structure of galactose oxidase. Here in CDO structure, Tyr-Cys cofactor plays a couple of totally different roles, including maintaining the stability of ferrous ion center, facilitating the cleavage of dioxygen bond during catalyzing reaction and preventing the formation of highly damaging free hydroxyl radical. Our proposed catalytic mechanism provides a base for future small molecule drug design and pathology study on CDO.
