Distinct Oxidoreductases Orchestrate Indolethiophene Skeleton Formation and <i>N</i> ‐Hydroxylation‐Mediated Bioactivation in Thienoxidolin Biosynthesis

Abstract

ABSTRACT 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.