Unexpected roles of aromatic ring-cleaving dioxygenases in oxygen-heterocycle degradation: Functional diversity, gene...

Oxygen-containing heterocycles (O-heterocycles), including tetrahydrofuran (THF) and 1,4-dioxane, are emerging water contaminants of growing concern because of their persistence and recalcitrance to conventional treatment processes. Biodegradation of these compounds requires ring cleavage, yet the enzymes responsible for this critical step have remained unidentified. By integrating affinity predictions with in vivo validation, we demonstrated that classical aromatic ring-cleaving dioxygenases, including the intradiol dioxygenases CatA1/CatA2 from Cupriavidus metallidurans ZM02 and the extradiol dioxygenase MhpB from Escherichia coli BL21, mediate the cellular cleavage of 2-hydroxytetrahydrofuran (2-OH THF), a key intermediate in THF metabolism, to yield 4-hydroxybutyric acid (GHB). This previously unrecognized activity occurs within the same substrate-binding pocket used for canonical aromatics, with residues E101 in CatA and H115/R212 in MhpB identified as key contributors to non-canonical heterocyclic cleavage through site-directed mutagenesis. Furthermore, catA genes are frequently redundant in bacteria degrading both O-heterocycles and aromatics, and their active sites are highly conserved, suggesting an evolutionary conserved strategy for coping with mixed pollutants. These findings expand the substrate spectrum of well-known dioxygenases to include O-heterocycles, establish a novel catalytic route for cyclic ether degradation in aquatic systems, and offer new opportunities for bioremediation and enzyme engineering in water treatment.