Jiramongkol Yannasittha, Patel Karishma, Johansen-Leete Jason, Maxwell Joshua W C, Chang Yiqun, Du Jonathan J, Passioura Toby, Cook Kristina M, Payne Richard J, White Mark D
School of Chemistry, The University of Sydney, Sydney, NSW, Australia.
Faculty of Science, Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia.
Nat Commun. 2025 May 22;16(1):4761. doi: 10.1038/s41467-025-59960-3.
N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O-dependent stability of proteins bearing an N-terminal cysteine residue through the N-degron pathway. Despite their important role in hypoxic adaptation, which renders them potential therapeutic and agrichemical targets, structural information on NCO substrate binding remains elusive. To overcome this challenge, we employed a unique strategy by which a cyclic peptide inhibitor of the mammalian NCO, 2-aminoethanethiol dioxygenase (ADO), was identified by mRNA display and used as a scaffold to graft substrate moieties. This allowed the determination of two substrate analogue-bound crystal structures of ADO. Key binding interactions were revealed, including bidentate coordination of the N-terminal residue at the metal cofactor. Subsequent structure guided mutagenesis identified aspartate-206 as an essential catalytic residue, playing a role in reactive oxygen intermediate orientation or stabilisation. These findings provide fundamental information on ADO substrate interactions, which can elucidate enzyme mechanism and act as a platform for chemical discovery.
N 端半胱氨酸氧化酶（NCOs）作为酶促氧（O）传感器，在动植物中协调细胞对缺氧的变化。它们通过 N-降解途径调节带有 N 端半胱氨酸残基的蛋白质的氧依赖性稳定性。尽管它们在缺氧适应中发挥着重要作用，使其成为潜在的治疗和农用化学品靶点，但关于 NCO 底物结合的结构信息仍然难以捉摸。为了克服这一挑战，我们采用了一种独特的策略，通过 mRNA 展示鉴定了哺乳动物 NCO 2-氨基乙硫醇双加氧酶（ADO）的环肽抑制剂，并将其用作嫁接底物部分的支架。这使得能够确定 ADO 的两种底物类似物结合晶体结构。揭示了关键的结合相互作用，包括金属辅因子处 N 端残基的双齿配位。随后的结构导向诱变确定天冬氨酸-206 是一个必需的催化残基，在活性氧中间体的定向或稳定中发挥作用。这些发现提供了关于 ADO 底物相互作用的基本信息，可阐明酶机制并作为化学发现的平台。
