Enzymatic Redox Gating Directs Oxidative Divergence in Acyclic Peroxides Biosynthesis

Peroxy natural products, including endoperoxides, acyclic peroxides, and hydroperoxides, are widely distributed across all domains of life, with many, such as artemisinin and prostaglandins, serving as clinically important agents. However, the enzymatic mechanisms by which nature installs O─O bonds have largely remained elusive. To date, only a limited number of endoperoxide-forming enzymes have been identified, while the enzymatic basis for acyclic peroxide assembly remains unknown. Here, we identify two homologous flavin-dependent enzymes, OxaJ and OtnJ, that catalyze enantioselective acyclic peroxide formation in the biosynthesis of oxanthromicin natural products. A conserved structural motif acts as a redox gate by blocking NADPH access to the active site, thereby promoting peroxide installation. Removal of this motif permits NADPH binding and redirects the enzyme's activity toward hydroxylation. This work establishes the first example of peroxide formation by a flavin-dependent enzyme and introduces redox gating as a previously unrecognized strategy for controlling oxidative divergence in enzymatic catalysis.