The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

中文翻译：

---

拟南芥legumainβ的结构和功能研究揭示了激活和底物识别的异构体特定机制。

液泡半胱氨酸蛋白酶legumain在种子成熟和植物程序性细胞死亡中起重要作用。由于它们的双重蛋白酶和连接酶活性，植物豆科菌素已成为具有特殊生物技术意义的物质，例如用于合成用于药物设计或蛋白质工程的环肽。然而，其双重蛋白酶和连接酶活性背后的分子机制仍知之甚少，限制了它们的应用。在这里，我们介绍拟南芥豆科菌种亚型β（AtLEGβ）在其酶原状态下的晶体结构。结合结构和生化实验，我们首次证明植物豆科植物蛋白编码不同的，同工型特异性激活机制。异构体γ（AtLEGγ）的自催化活化受赋予潜伏期的二聚体状态控制，单体AtLEGβ的激活与浓度无关。另外，在AtLEGβ中，植物特征性的两链中间状态通过疏水作用而不是离子相互作用来稳定，就像在AtLEGγ中一样，从而导致了明显不同的pH稳定性曲线。AtLEGβ的晶体结构显示出不受限制的非底物底物结合口袋，与广泛的底物特异性一致，这是通过降解组学测定确定的。除其蛋白酶活性外，我们还显示AtLEGβ表现出真正的肽连接酶活性。尽管已经报道了其他植物豆科植物的裂解依赖性转肽酶活性，但是AtLEGβ是能够连接游离末端的植物豆科植物的第一个实例。