Updated project metadata. Ubiquitin-like protein activator 4 (Uba4) is required for the thiolation of uridine bases in eukaryotic transfer RNA. Uba4 catalyzes the sequential adenylation and thiocarboxylation of the C-terminus of Ubiquitin related modifier 1 (Urm1), acting as a hybrid between an E1-like activating enzyme and a sulfur-transferase. Structural and mechanistic details of the eukaryotic Uba4 protein and its respective reaction intermediates remained elusive. Here, we report the high-resolution crystal structures of full length Uba4 from Chaetomium thermophilum and its heterodimeric complex with Urm1. The structures show, how the aligned adenylation and rhodanese-like domains of Uba4 orchestrate substrate binding and specifically recognize and activate Urm1 at its C-terminus. We use complementary assays in vitro and in vivo to functionally validate all our structural findings. Furthermore, we uncover that under oxidative stress the specific relay of catalytic cysteines, similar to E1-E2 cascades, protects Uba4 from covalent conjugation by its own product, namely thiocarboxylated Urm1.