Structural and Mechanistic Insights into Azetidine-associated αKG-NHFe Enzyme OkaE with Multifunctional Catalysis

α-Ketoglutarate-dependent mononuclear non-haem iron (αKG-NHFe) enzymes exhibit remarkable catalytic versatility. To date, OkaE is the only αKG-NHFe enzyme capable of synthesizing a pharmacophoric motif azetidine ring via unique C–C bond formation. Our investigation reveals the unexpected multifunctionality of OkaE, which enables not only azetidine formation but also scaffold diversification through a sequential oxidations, including hydroxylation, epoxidation, and ring cleavage. Isotopic labelling studies reveal that a second O₂ molecule participates in sequential epoxidation and ring cleavage, leading to the incorporation of two oxygen atoms into product 7 within a single catalytic cycle. High-resolution crystal structures of OkaE•CoII•αKG•okaramine A complex unveil a non-canonical cis-serine conformation within a b-hairpin that stabilizes substrate binding. Additionally, a unique Methionine–π interaction network involving residues M64-M71-W79-M122 facilitates the binding of the indole tail (A- and B-rings) of the substrate. Alanine scanning and crystallographic analysis suggest that this interaction network fine-tunes the spatial orientation of the substrate relative to the active metallo-centre, thereby activating distinct reaction pathways at the C8a or OH3a positions. In addition, QM/MM simulations reveal that the catalytic cycle is initiated by a dynamic rotation of the FeIV=O species from a distal-type αKG binding mode, enabling bifurcation of the reaction pathway. This study elucidates the structural and mechanistic basis of OkaE’s reactivity and establishes it as a programmable biocatalyst for the post-modification and diversification of natural products.