Cardiac glycosides are secondary metabolites that have evolved independently in at least 12 plant families to serve as an herbivore defense mechanism. While cardiac glycosides are widely used by humans as a treatment for heart arrhythmias and heart failure, their biosynthetic pathway remains unknown. Cardenolides are cardiac glycosides that have a 6/6/6/5 steroid core with a five-membered lactone ring and one or more sugars. We used Erysimum cheiranthoides (wormseed wallflower) as a model system to identify UDP-glycosyltransferases (UGTs) that add a sugar to the aglycone steroid core. We have identified candidate UGT genes based on shared homology with UGTs known to glycosylate steroids in other plants. Because many putative substrates are commercially unavailable, we have developed a novel semi-targeted mass spectrometry screening system to identify the substrates of our candidate UGTs by incubating the purified proteins with plant extracts. This approach has allowed us to successfully identify the substrates of several candidate UGTs. Next, we will confirm the activity of the candidate UGTs by generating knockouts of the lead UGT gene candidates in E. cheiranthoides plants. The current system can then be used to identify additional genes of interest in the cardenolide biosynthetic pathway. Methods that we have developed for rapidly identifying substrates of UGTs involved in specialized metabolism may also be applied to other plant systems to allow for the characterization of enzymes involved in a diverse range of biosynthetic processes.
