In this study, we demonstrate that PolF, a member of the haem-oxygenase-like dimetal oxidase/oxygenase (HDO) superfamily, catalyzes the conversion of L-isoleucine (L-Ile) and L-valine into their azetidine derivatives via a 3,4-desaturated intermediate. Mechanistic analyses reveal that a μ-peroxo-Fe(III)2 intermediate mediates the cleavage of unactivated C–H bonds, while subsequent reactions—including C–N bond formation—likely proceed through radical pathways. Additionally, we identify PolE, a DUF6421 family enzyme, as an Fe- and pterin-dependent oxidase that promotes L-Ile desaturation, thereby enhancing substrate flux for PolF. Collectively, these findings shed light on azetidine biosynthesis and expand our understanding of HDO enzyme catalysis. The significance of our work was also highlighted in the news section of the School of Medicine.
Department of Biochemistry, Duke University School of Medicine
