Discovery of corynanthe synthases in Camptotheca acuminata: medium-chain dehydrogenase/reductase-based gateway to corynanthe-type compound diversification.

Main conclusion: We combined bioinformatic analysis with in vitro biochemical assays to identify and characterize six medium-chain dehydrogenase/reductase (MDR) enzymes involved in corynanthe alkaloid biosynthesis in Camptotheca acuminata. Camptotheca acuminata, commonly known as the happy tree, is a medicinal plant from the monoterpenoid indole alkaloids (MIAs)-rich Nyssaceae family. In this study, we combined bioinformatic analysis and biochemical investigations to identify new enzymes involved in the biosynthesis of corynanthe alkaloids, an MIA subgroup, in C. acuminata. We identified six medium-chain dehydrogenase/reductase (MDR) enzymes, four of which catalyze the formation of diverse simple corynanthe alkaloids. Structural modeling of the two newly identified enzymes, namely CaMDR1 and CaMDR2, revealed key active-site residues Ser52 and Ser55/His55, crucial for their product profiles. The identification of these new corynanthe synthases further advances our understanding of the formation and diversification of corynanthe alkaloids and MIAs in happy tree and related species.