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

Abstract

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•Co^II•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 Fe^IV=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.