Discovery and Mechanism of a Diiron Enzyme in Ethylidene Azetidine Formation

Azetidine is a strained four-membered <i>N</i>-heterocycle that has important applications in medicinal chemistry and organic synthesis. Despite its relevance to human health and agriculture, azetidine biosynthesis remains largely unexplored. Herein, the DUF6202 family enzyme PolF is shown to utilize a diiron center and O₂ to catalyze sequential desaturation and azetidination of l-isoleucine to yield the ethylidene azetidine moiety during the biosynthesis of polyoxin. Crystallographic analysis of PolF in complex with iron (or manganese) and l-isoleucine (or an alkene intermediate) reveals that it is a heme oxygenase-like diiron oxidase with a new iron-binding motif. Trapping an O₂-bound intermediate <i>in crystallo</i> along with a distinctive iron-to-protein ratio observed under anoxic and aerobic environments indicates that binding of the second iron occurs in an O₂-dependent manner. Mössbauer and stopped-flow optical absorption spectroscopy together with deuterium kinetic isotope effect measurements demonstrate that a μ-peroxodiiron(III) species is likely responsible for C-H bond activation. Mass Spectrometry analysis reveals that the decay of the μ-peroxodiiron(III) species is accompanied by the formation of a desaturated intermediate. Bioinformatic analysis identifies <i>ca.</i> 200 PolF homologues indicative of new biosynthetic pathways for azetidine-containing natural products.