In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

在高等植物中，对外源性缺氧的分子反应是由第七组乙烯反应因子（ERF-VII）驱动的。这些转录调节因子在缺氧条件下在细胞核中积累，以激活厌氧基因，但在含氧量正常的条件下，通过氧敏感植物半胱氨酸氧化酶 (PCO) 的作用而不稳定。 PCO 催化氧与 ERF-VII 的保守 N 末端半胱氨酸反应形成半胱氨酸亚磺酸，通过 N-降解决定子途径的 Cys/Arg 分支触发降解。因此，PCO 是植物氧信号系统的重要组成部分，将环境刺激与细胞和生理反应联系起来。合理控制 PCO 活性可以调节 ERF-VII 水平并提高抗洪能力，但需要详细的结构信息。我们报道了拟南芥组成型表达的 PCO4 和 PCO5 的晶体结构，分辨率分别为 1.24 和 1.91 Å。这些结构表明，PCO 包含一个类似 cupin 的支架，该支架支持由三个组氨酸协调的中心金属辅因子。虽然这种整体结构与其他硫醇双加氧酶一致，但对活性位点的仔细检查表明其他催化特征并不保守，这表明 PCO 可能使用不同的机制来氧化其底物。通过酵母和植物互补测定，两个活性位点残基的保守取代对体外和体内 PCO4 功能都有显着影响。总的来说，我们的数据确定了 PCO 活性所需的关键结构元素，并为工程作物提供了一个平台，以提高耐缺氧能力。