NAE1/UBA3-UBE2M are E1 and E2 Enzymes for the Urm1 Modification in Human Cells

In eukaryotes, it serves dual roles as a sulfur donor for tRNA modification and a posttranslational protein modifier. Urm1 is proposed to be a primitive protein modifier and a potential precursor to the more complex ubiquitin system.

However, no specific activating enzyme (E1), conjugating enzyme (E2), or ligase (E3) has been reported for the Urm1 modification cascade in human cells. In this study, we design an activity-based Urm1 probe to covalently capture cysteine enzymes functioning in the Urm1 signaling pathway. Through proteomic characterization and cell-based validation, we identify NAE1/UBA3 and UBE2M as E1 and E2 enzymes, respectively, for the urmylation pathway under both normal and oxidative stress conditions.