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.

Mycobacteriophages-derived lysis proteins such as LysB are promising antibiotic alternatives to counter drug-resistant pathogenic mycobacteria such as Mycobacterium tuberculosis and nontuberculous mycobacteria. Previous work has correlated the antimycobacterial activity of LysB with its peptidoglycan-binding domain (PGBD) in the N-terminal domain (NTD), but underlying mechanistic is lacking. Here, we discovered that NTD is crucial for the antimycobacterial activity of TM4LysB due to the likely effect on TM4LysB's translocation to its cognate substrate mycolyl-arabinogalactan-peptidoglycan (mAGP), but neither the effect on NTD's PG-binding properties nor TM4LysB's catalytic activity. We also found that the antimycobacterial activity of several different LysBs, including TM4LysB, D29LysB and Ms6LysB, is strongly influenced by a highly conserved super-secondary structure (SSS) in the catalytic domain which structurally distinguishes LysBs from members of the cutinase family and allows PG-binding ability of catalytic domain. The SSS is essential for both catalytic activity and probably efficient translocation of LysBs to the mAGP layer.

Met1 type ubiquitination and deubiquitination are involved in the regulation of many fundamental processes such as inflammation and innate immunity, and their interference by pathogens can suppress immune responses in human cells. However, no plant-derived deubiquitinases (DUBs) against Met1 ubiquitin chains have been reported. Using a dehydroalanine (DHA)-bearing Met1 diubiquitin (Met1-diUb) suicide probe, synthesized in one-pot, we identified OTUB1 from Oryza sativa (OsOTUB1) and uncovered its preference for Met1 ubiquitin chains. Also, by resolving the apo structure of OsOTUB1 and its complex with Ub or Met1-diUb, we demonstrated that OsOTUB1 hydrolyses Met1 ubiquitin chains by activation of both the distal and proximal ubiquitin, which is different from OTULIN and expands our mechanistic understanding of the DUB-mediated hydrolysis of Met1 ubiquitin chains. Through large-scale sequence alignment and hydrolysis experiments, two sites in the S1' pocket of the OTUB subfamily (OTUBs) were found to determine the hydrolytic ability of OTUBs against Met1 ubiquitin chains, regardless of species. Furthermore, by analyzing the species distribution of OTUBs capable of hydrolyzing Met1 ubiquitin chains, we found that whereas this activity does not exist in metazoans, it is conserved in green plants (Viridiplantae). This discovery may inform studies of the differentiation between primitive plants and animals.