Evidence of an activity-enhancing conformational shift in Arabidopsis thaliana Plant Cysteine Oxidase 4 induced by binding of substrate or substrate-mimics.

Plant cysteine oxidases (PCOs) are a family of O2-dependent, thiol dioxygenase enzymes that help to coordinate plant responses to flooding by determining the stability of hypoxia-related transcription factors, Group VII Ethylene Response Factors (ERF-VIIs). Under normoxia, PCOs use O2 to catalyse the oxidation of the Nt-Cys of ERF-VIIs. The resultant N-degron proceeds along the Cys/Arg N-degron pathway for degradation. Conversely, hypoxic conditions such as those experienced during flooding, decrease PCO activity, stabilising ERF-VIIs which proceed to upregulate hypoxia responsive genes, enabling adaptations to submergence. PCOs are a target for improving plant flood tolerance. Inhibition of PCOs may prepare plants for submergence by promoting upregulation of hypoxia responsive genes. Previous work identified small molecule inhibitors of AtPCO4 and demonstrated that their use as a pre-treatment improved seedling tolerance to subsequent anoxia exposure. In this work, the pursuit of a peptide-based inhibition approach led to the development of ERF-VII derived peptidomimetics with modified N-termini. Upon testing in vitro, the peptidomimetics unexpectedly enhanced, rather than inhibited, AtPCO4 activity. Furthermore, anaerobic preincubation of PCOs with substrate itself was found to induce a similar response. Hydrogen-deuterium exchange mass spectrometry indicated that preincubation with peptidomimetic induces a conformational change in PCO structure. This suggests that substrate binding under anaerobic conditions could promote "conformational priming" of AtPCO4, where flexible regions within or surrounding the active site adopt a conformation that favours enhanced enzymatic activity. These data are the first evidence for dynamic movement of PCO structures and may be of significance for post-hypoxia PCO activity in planta.