Biochemical characterisation of a novel mammalian oxygen sensing enzyme

Abstract

Oxygen (O2) homeostasis is an essential biological process that regulates O2 supply and demand to alleviate hypoxic stress. At molecular level, O2 concentrations are monitored by enzymatic O2 sensors which couple O2 availability to cellular activity through O2-dependent post-translational modifications. Recently, 2-aminoethanethiol dioxygenase (ADO) is identified with ability to sense and respond to O2 fluctuations. ADO catalyses target Nt-Cys sulfinylation in normoxia, promoting Ntarginylation, ubiquitination and proteasomal degradation in N-degron pathway. ADO is inactivated during hypoxia due to its low affinity for O2, resulting in substrate stabilisation and cellular changes. Despite this important role in O2 adaptation, little is known about its substrate binding requirements and underlying biochemistry. To address the knowledge gaps, this thesis analysed key substrate interactions using complementary techniques and established mRNA display-derived inhibitors of ADO. SPR and enzymatic assays demonstrate that free Nt-thiol and Nt-amine are crucial for substrate engagement, with residues immediately next to Nt-Cys moderately influencing ADO binding and activity. Cyclic peptide inhibitors of ADO were identified, characterised and used as a scaffold to elucidate the first substrate (analogue) bound crystal structure of ADO. This revealed bidentate coordination of the Nt-residue, leaving one ligation site on the metal cofactor available for O2 activation. It also highlighted key active site residues involved in substrate binding and activity, including Asp206, a catalytically essential amino acid with a putative role in substrate binding and product release. Together, this work establishes key features involved in ADO substrate binding and turnover, describes the first selective inhibitors of ADO, and defines fundamental active site interactions, which will help identify new targets, aid therapeutic discovery, and illuminate enzyme mechanisms, respectively.