Rieske Oxygenase-Catalyzed Oxidative Late-Stage Functionalization during Complex Antifungal Polyketide Biosynthesis.

Rieske oxygenases (ROs) from natural product biosynthetic pathways are a poorly studied group of enzymes with significant potential as oxidative functionalization biocatalysts. A study on the ROs JerL, JerP, and AmbP from the biosynthetic pathways of jerangolid A and ambruticin VS-3 is described. Their activity was successfully reconstituted using whole-cell bioconversion systems coexpressing the ROs and their respective natural flavin-dependent reductase (FDR) partners. Feeding authentic biosynthetic intermediates and synthetic surrogates to these strains confirmed the involvement of the ROs in hydroxymethylpyrone and dihydropyran formation and revealed crucial information about the RO's substrate specificity. The pronounced dependence of JerL and JerP on the presence of a methylenolether allowed the precise temporal assignment of RO catalysis to the ultimate steps of jerangolid biosynthesis. JerP and AmbP stand out among the biosynthetic ROs studied so far for their ability to catalyze clean tetrahydropyran desaturation without further functionalizing the formed electron-rich double bonds. This work highlights the remarkable ability of ROs to highly selectively oxidize complex molecular scaffolds.