α-Ketoglutarate-dependent mononuclear non-haem iron (αKG-NHFe) enzymes exhibit remarkable catalytic versatility. Here, we report that OkaE, previously known for azetidine formation, displays unexpected multifunctionality, enabling sequential hydroxylation, epoxidation, and ring cleavage. Isotopic labelling studies revealed that a second O2 molecule is incorporated within a single catalytic cycle. High-resolution crystal structures of the OkaE•CoII•αKG•okaramine A complex unveiled that a non-canonical cis-serine conformation within a β-hairpin and a unique methionine–π interaction network stabilized substrate binding. Mutagenesis and crystallographic analysis suggested that this network governs the spatial orientation of the substrate relative to the metallo-centre, thereby activating distinct reaction pathways at the 3a-OH or C8a positions. Furthermore, QM/MM simulations indicated that the catalytic cycle involved dynamic rotation of the FeIV=O species from a distal-type αKG binding mode. These findings elucidate the mechanistic basis of OkaE reactivity, highlighting its potential as a programmable biocatalyst for natural product diversification.
Junjie Yu (Sun,) studied this question.