Algal kainoid synthases exhibit substrate-dependent hydroxylation and cyclization activities

Abstract

FeII/a-ketoglutarate-dependent dioxygenases (Fe/aKG) are a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/aKG 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 β-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 substratedependent chemistry improves our understanding of Fe/aKG enzymes and their biocatalytic application.