An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase

N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O₂) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O₂-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.