Abstract
Monoterpene indole alkaloids (MIAs) are a large, structurally diverse class of bioactive natural products. These compounds are biosynthetically derived from a stereoselective Pictet-Spengler condensation that generates a tetrahydro-β-carboline scaffold characterized by a 3 S stereocenter. However, a subset of MIAs contain a non-canonical 3 R stereocenter. Herein, we report the basis for 3 R -MIA biosynthesis in Mitragyna speciosa (Kratom). We discover the presence of the iminium species, 20 S -3-dehydrocorynantheidine, which led us to hypothesize that isomerization of 3 S to 3 R occurs by oxidation and stereoselective reduction downstream of the initial Pictet-Spengler condensation. Isotopologue feeding experiments implicated young leaves and stems as the sites for pathway biosynthesis, facilitating the identification of an oxidase/reductase pair that catalyzes this epimerization. This enzyme pair has broad substrate specificity, suggesting that the oxidase and reductase may be responsible for the formation of many 3 R -MIAs and downstream spirooxindole alkaloids in Kratom. These enzymes allow biocatalytic access to a range of previously inaccessible pharmacologically active compounds.