Oxidative bicyclic ring system formation involving indole diterpene biosynthesis: Remarkable substrate tolerance of a prenyltransferase and flavoprotein oxidase

Oxidative modifications are key biosynthetic processes responsible for expanding the structural diversity of indole diterpenes (IDTs). In this study, we focused on the function of prenyltransferases and flavoprotein oxidases for the synthesis of nodulisporic acid-type and shearinine-type bicyclic rings found on the indole moiety. An in vitro enzymatic reaction of a prenyltransferase JanD, which mediates diprenylation at the C5- and C6-positions, revealed that JanD recognizes a C13‑hydroxy group on the diterpene moiety. Subsequent enzymatic reactions with flavoprotein oxidases, JanO, and NodO, indicated that JanO can accommodate penta/hexacyclic IDTs, while NodO exhibits strict substrate specificity toward pentacyclic IDTs. The broad substrate tolerance of JanD and JanO provides opportunities for the synthesis of unnatural IDTs based on artificial reconstitution in a heterologous host.