Expression, purification, and biochemical characterization of micro- and macroalgal kainoid synthases

ABSTRACT

Kainoid natural products are a series of potent ionotropic glutamate receptor agonists produced by a variety of divergent marine micro- and macro-algae. The key biosynthetic step in the construction of the pyrrolidine ring pharmacophore involves a unique branch of non-heme iron α-ketoglutarate dependent dioxygenases (Fe/αKGs) termed the kainoid synthases. These Fe/αKG homologs catalyze a stereoselective C-H abstraction followed by a radical carbon-carbon bond reaction to form the bioactive core on N-prenylated L-glutamic acid substrates. In this article, we describe the expression, purification, and biochemical characterization of four divergent kainoid synthases (DabC, RadC1, DsKabC, GfKabC). Furthermore, we compare and contrast their substrate preferences and product distributions, and provide some preliminary insight into how to repurpose these enzymes for whole cell biocatalysis.