Characterization of Late Acting Enzymes in Neuroactive Lycopodium Alkaloid (HupA) Biosynthesis

Plants have evolved to produce many diverse small molecules that affect mammalian nervous systems, making them a rich resource for treatments for neurological disorders. A prominent example is lysine-derived alkaloids, which are one of the least understood class of metabolites in plant biosynthesis. To better understand how plants synthesize these metabolites, we studied the biosynthesis of Lycopodium alkaloids that are produced by club mosses, focusing on Huperzine A (HupA)—a notable example of Lycopodium alkaloids known for its potential treatment for neurodegenerative diseases like Alzheimer’s disease due to its acetylcholinesterase inhibitory effect. Through combination of metabolomics, transcriptomics, and enzymatic logics, we identified two novel 2-oxoglutarate dependent dioxygenases (2OGDs), non-heme Fe(II) bound oxidoreductase enzymes, in the late-biosynthetic pathway of HupA that do uncommon oxidative reactions—oxidative ring cleavage of HupB with a production of terminal olefin of HupC and irreversible double bond isomerization of HupC to HupA. To further study these remarkable novel chemistries performed by 2OGDs, the mechanistic details of these two 2OGDs were characterized in vitro. The first 2OGD alone was found to perform both oxidative ring cleavage and desaturation to form HupC from HupB through canonical catalytic cycle of 2OGD. The second 2OGD performed redox-neutral double bond isomerization on HupC to produce HupA using stoichiometric amount of 2-oxoglutarate (2OG). These mechanistic studies of ours on 2OGDs in the downstream of HupA biosynthesis expand the diversity of the known chemical transformations catalyzed by 2OGDs and offer deeper insights into how 2OGDs can perform complex chemistries, which could also potentially assist in identification and understanding of future enzymes involved in the biosynthesis of other lysine-derived bioactive alkaloids found in plants.