Bifunctional P450 Catalyzes Sequential Hydroxylation and Macrocyclization in Emestrin A Biosynthesis

Emestrin A, an epipolythiodioxopiperazine with a rare 15-membered ether–lactone macrocycle, exhibits significant antitumor activity. However, the late-stage enzymatic steps that establish and modify this scaffold and how these modifications influence bioactivity remain unclear. Here, targeted gene deletions in Aspergillus nidulans A6 revealed three P450 monooxygenases involved in oxidative tailoring. Among them, EmeQ was identified as a bifunctional P450 that catalyzes aromatic hydroxylation and phenol coupling, the latter of which drives the macrocyclization of the ether–lactone ring. Computational analysis delineated the coupling mechanism, and structure-guided mutagenesis successfully disentangled the dual reactivity, shedding light on how EmeQ coordinates the two sequential steps. In parallel, cytotoxicity assays demonstrated that hydroxyl groups introduced by late-stage tailoring are important for activity. This work provides a mechanistic elucidation of emestrin late-stage biosynthesis and establishes EmeQ as both a hydroxylase and a macrocyclase central to scaffold construction. The natural and engineered mutant enzymes characterized here expand the toolbox for the chemoenzymatic synthesis and synthetic biology of emestrins.