Algal kainoid synthases exhibit substrate-dependent hydroxylation and cyclization activities

FeII/-ketoglutarate-dependent dioxygenases (Fe/KG) are a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/KG homologs catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis. However, this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over {beta}-hydroxylation, with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologs and provided insight into substrate recognition and reactivity trends. Investigation of this substrate-dependent chemistry improves our understanding of Fe/KG enzymes and their biocatalytic application.