Younkin, G.; Holland, C. K.; Jander, G.

>Cardenolide biosynthesis evolved convergently in several plant lineages, including wallflowers (Erysimum, Brassicaceae). Although the first steps of the biosynthetic pathway, which involve conversion of sterols to pregnane derivatives, have been characterized in Erysimum cheiranthoides and other species, key enzymes that catalyze the 14{beta}- and 21-hydroxylation of the steroid core remained elusive. >In this study, we used comparative transcriptomic analysis of different Erysimum species to identify 2-oxoglutarate dependent dioxygenases (2OGDs) that catalyze these reactions in E. cheiranthoides: Erche03g034150 (CARDENOLIDE METABOLISM 5; CARD5), which is related to ALKENYL HYDROXYALKYL PRODUCING 1 (AOP1), and Erche01g020322 (CARDENOLIDE METABOLISM 6; CARD6), which arose from a duplication of DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1). >Knockout mutants of both genes are deficient in cardenolide biosynthesis and instead accumulate pathway intermediates. Based on transient expression and activity in Nicotiana benthamiana, and we identified CARD5 as the likely 14{beta}-hydroxylase, and CARD6 as the 21-oxygenase. Finally, enzyme modeling and substrate docking identified key residues that may allow shifts to substrate recognition during neofunctionalization. >The results of this research provide new insight into the evolution of cardenolide biosynthesis and have potential practical applications in the engineering of steroid-derived compounds for medical uses.