Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1’ pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.

线性（Met1 连接）泛素化涉及炎症和先天免疫信号传导。以前的研究已经表征了在哺乳动物系统中调节这种修饰的添加和去除的酶。然而，仅报道了少数靶向 Met1 连接的泛素链的植物来源的去泛素酶，其作用机制仍然难以捉摸。在这里，我们使用带有脱氢丙氨酸的 Met1-二泛素自杀探针，发现了来自Oryza sativa的 OTUB1(OsOTUB1) 作为 Met1 连接的泛素链靶向去泛素酶。通过解析 apo OsOTUB1 和 OsOTUB1/Met1-二泛素复合物的晶体结构，我们发现 Met1 活性是由 OsOTUB1 S1' 口袋中的 Met1 特异性基序赋予的。大规模序列比对和水解实验证明这些基序是跨物种 OTUB 亚科中 Met1 活性的一般决定因素。对能够水解 Met1 连接的泛素链的 OTUB 的物种分布分析表明，这种活性在绿色植物 ( Viridiplantae ) 中是保守的，在后生动物中不存在，为了解原始植物和动物之间的进化分化提供了见解。