A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis

Oxygen heterocycles—in particular, tetrahydropyrans (THPs) and tetrahydrofurans—are common structural features of many biologically active polyketide natural products. Mupirocin is a clinically important antibiotic isolated from Pseudomonas fluorescens and is assembled on a THP ring, which is essential for bioactivity. However, the biosynthesis of this moiety has remained elusive. Here, we show an oxidative enzyme-catalysed cascade that generates the THP ring of mupirocin. Rieske non-haem oxygenase (MupW)-catalysed selective oxidation of the C8–C16 single bond in a complex acyclic precursor is combined with an epoxide hydrolase (MupZ) to catalyse the subsequent regioselective ring formation to give the hydroxylated THP. In the absence of MupZ, a five-membered tetrahydrofuran ring is isolated, and model studies are consistent with cyclization occurring via an epoxide intermediate. High-resolution X-ray crystallographic studies, molecular modelling and mutagenesis experiments of MupZ provide insights into THP ring formation proceeding via an anti-Baldwin 6-endo-tet cyclization.