Distinct Oxidoreductases Orchestrate Indolethiophene Skeleton Formation and N-Hydroxylation-Mediated Bioactivation in Thienoxidolin Biosynthesis.

Microbial sulfur-containing secondary metabolite thienodolin (1) features a unique tricyclic thieno[2,3-b]indole scaffold, yet its biosynthesis has remained enigmatic. Here, we uncover an unexpected enzymatic logic in which a consortium of distinct oxidoreductases cooperatively orchestrates indolethiophene skeleton formation and subsequent bioactivation, ultimately generating the authentic antibacterial metabolite thienoxidolin (10). Following thiotryptophan formation by SDR enzyme TndE, the heme-dependent DUF6875 enzyme TndD initiates C-S bond formation via N-hydroxylation to yield a dearomatized tricyclic species, which is efficiently driven forward and stabilized by aromatization catalyzed by the FAD-dependent oxidoreductase TndG. Notably, TndD functions as a bidirectional redox enzyme, reverting the N-hydroxyl group to the stable N-H form to complete the indolethiophene scaffold construction. After amide formation, late-stage N-hydroxylation by the cytochrome P450 enzyme TndC, previously misassigned as the C-S bond-forming enzyme, produces the bioactive product 10. Intriguingly, TndD may also mediate the deactivation of 10 back to 1, representing an intrinsic self-protection mechanism. Together, these results expand the catalytic repertoire of heme-dependent enzymes and highlight reversible N-hydroxylation as a pivotal strategy for heterocycle formation and bioactivity regulation in microbial secondary metabolism.