Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O2 as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure-function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.

中文翻译：

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人半胱胺双加氧酶的晶体结构为其作为氧传感器的功能提供了结构原理。

半胱胺双加氧酶 (ADO) 通过使用 O2 作为共底物将半胱胺和 N 端含半胱氨酸蛋白质的硫氧化为相应的亚磺酸，在调节硫醇代谢和维持人体氧稳态方面发挥着至关重要的作用。然而，作为唯一同时处理小分子和蛋白质底物的硫醇双加氧酶，ADO 如何处理不同大小的不同底物以实现各种反应尚不清楚。知识差距主要是由于未解决三维结构，因为 ADO 无法与其他已知的硫醇双加氧酶直接比较。在此，我们报告了人类 ADO 的第一个晶体结构，分辨率为 1.78 Å，具有镍结合金属中心。结晶是通过金属取代和 C18S/C239S 双突变实现的。金属中心位于靠近入口处的隧道中，两侧是环。虽然 ADO 似乎使用广泛的灵活性来处理不同大小的底物，但它也使用脯氨酸和脯氨酸对来维持核心蛋白质结构并保留对催化至关重要的残基。这一特征将 ADO 与仅氧化小分子底物的硫醇双加氧酶区分开来，这可能解释了其不同的底物特异性。我们的研究结果还通过修饰 N-degron 底物来转导对缺氧的反应，阐明了 ADO 作为氧传感器的结构基础。因此，这项工作填补了硫醇双加氧酶家族结构-功能关系的空白，并为进一步针对氧传感受损的机制研究和治疗干预提供了平台。