Structural and mechanistic insights into azetidine-associated αKG-NHFe enzyme OkaE with multifunctional catalysis

α-Ketoglutarate-dependent mononuclear non-haem iron (αKG-NHFe) enzymes are catalytically versatile, yet OkaE is unique for synthesizing azetidine rings via C–C bond formation. Here, we report the unexpected multifunctionality of OkaE, which catalyzes sequential oxidations. Isotopic labelling studies demonstrate that a second O₂ molecule participates in sequential epoxidation and ring cleavage, incorporating two oxygen atoms within a single catalytic cycle to form the previously unknown structure, neuokaramine IV. Crystal structures of the OkaE•CoII•αKG•okaramine A complex unveil a unique methionine–π interaction network that facilitates substrate binding. Mutational and crystallographic analyses suggest this network fine-tunes substrate orientation relative to the metallo-centre, activating distinct reaction pathways at the 3a-OH or C8a positions. QM/MM simulations indicate that dynamic rotation of the FeIV=O species initiates the cycle, enabling reaction bifurcation. This study elucidates the structural and mechanistic basis of OkaE’s reactivity, highlighting its potential as a programmable biocatalyst for natural product diversification. OkaE is an αKG-NHFe enzyme capable of synthesizing the pharmacophoric motif azetidine ring via unique C–C bond formation. Here, the authors report the multifunctionality of OkaE, which enables not only azetidine formation but also scaffold diversification through sequential oxidations, including hydroxylation, epoxidation, and ring cleavage.