Gunawardana Dona M, Southern Daisy A, Flashman Emily
Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.
Department of Biology, University of Oxford, Oxford, OX1 3RB, UK.
Sci Rep. 2024 Dec 30;14(1):31960. doi: 10.1038/s41598-024-83508-y.
Plant Cysteine Oxidases (PCOs) are oxygen-sensing enyzmes that catalyse oxidation of cysteinyl residues at the N-termini of target proteins, triggering their degradation via the N-degron pathway. PCO oxygen sensitivity means that in low oxygen conditions (hypoxia), their activity reduces and target proteins are stabilised. PCO substrates include Group VII Ethylene Response Factors (ERFVIIs) involved in adaptive responses to the acute hypoxia experienced upon plant submergence, as well as Little Zipper 2 (ZPR2) and Vernalisation 2 (VRN2) which are involved in developmental processes in hypoxic niches. The PCOs are potential targets for improving submergence tolerance through enzyme engineering or chemical treatment. To achieve this, a detailed understanding of their biological function is required. Here, we report development of an assay that exploits the intrinsic fluorescence of Arabidopsis thaliana PCO tryptophan residues. By using Ni(II)-substitued enzymes and preparing the assay under anaerobic conditions, tryptophan fluorescence quenching is observed on enzyme:substrate complex formation, allowing quantification of binding affinities. Our assay revealed that, broadly, AtPCO4 and AtPCO5 have stronger interactions with ERFVII substrates than ZPR2 and VRN2, suggesting ERFVIIs are primary targets of these enzymes. It also revealed a positive cooperative binding effect for interactions between AtPCOs4/5 and ERFVIIs and ZPR2. The assay is experimentally straightforward and can be used to further interogate PCO interactions with substrates.
植物半胱氨酸氧化酶（PCOs）是氧感应酶，可催化靶蛋白N端半胱氨酰残基的氧化，通过N-端规则途径触发其降解。PCO的氧敏感性意味着在低氧条件下（缺氧），它们的活性降低，靶蛋白得以稳定。PCO的底物包括参与植物淹水时急性缺氧适应性反应的VII族乙烯反应因子（ERFVIIs），以及参与缺氧生态位发育过程的小拉链2（ZPR2）和春化2（VRN2）。PCOs是通过酶工程或化学处理提高淹水耐受性的潜在靶点。要实现这一点，需要详细了解它们的生物学功能。在此，我们报告了一种利用拟南芥PCO色氨酸残基固有荧光的检测方法的开发。通过使用镍（II）取代的酶并在厌氧条件下进行检测，在酶与底物复合物形成时观察到色氨酸荧光猝灭，从而能够对结合亲和力进行定量。我们的检测表明，总体而言，AtPCO4和AtPCO5与ERFVII底物的相互作用比与ZPR2和VRN2更强，这表明ERFVIIs是这些酶的主要靶点。它还揭示了AtPCOs4/5与ERFVIIs和ZPR2之间相互作用的正协同结合效应。该检测在实验上很简单，可用于进一步探究PCO与底物的相互作用。
