Reverse-prenylated phenolic compounds are an abundant class of bioactive plant natural products. Hyperixanthone A, an inhibitor of multidrug-resistant Staphylococcus aureus, is a polyprenylated xanthone carrying
two forward geminal and one reverse prenyl group. Although prenyltransferases responsible for the forward
prenylations were identified, the final reverse prenylation reaction remained elusive. No plant enzyme catalyzing reverse prenylation of an aromatic carbon has been described so far. Here we use metabolic profiling
and transcriptomic information from Hypericum perforatum and H. sampsonii to identify homologous
enzymes involved in the formation of reverse-prenylated xanthones and characterize their functions using
in vitro, in vivo, and in silico approaches. The identified enzymes are non-canonical UbiA-type prenyltransferases, which surprisingly catalyze both forward and reverse prenylations with different regioselectivities.
Reconstruction of the enzyme cascade in Saccharomyces cerevisiae and Nicotiana benthamiana confirmed
reverse-prenylated hyperixanthone A as the major product. Molecular modeling and docking simulations
supported by site-directed mutagenesis suggest two distinct binding modes, which enable forward and
reverse prenylations and provide a rationale for the preferred catalysis of the reverse prenyl transfer reaction. The identification of reverse prenylation augments the repertoire of reactions catalyzed by
membrane-bound UbiA-type plant aromatic prenyltransferases. The insights also provide a new tool for the
biotechnological modification of pharmaceutically valuable natural products.