Biosynthetic Enzymes in the Assembly and Derivatization of Paralytic Shellfish Toxins

Abstract

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by freshwater cyanobacteria and marine dinoflagellates. These natural toxins are implicated in harmful algal blooms, toxic red tides, and paralytic shellfish poisoning. Despite their notoriety as biological toxins, PSTs have been indispensable tools for studying the structure and function of mammalian voltage-gated sodium channels (VGSCs) for several decades. PSTs share a tricyclic scaffold that contains two cyclic guanidinium ions that is differentially functionalized, augmenting the molecules’ affinity for VGSCs. This tunable toxicity poses an attractive avenue for the development of therapeutics targeting VGSCs; however, the synthesis and selective functionalization of this complex scaffold is synthetically challenging. The enzymes responsible for the assembly and derivatization of PSTs have the potential to be valuable tools for the chemoenzymatic synthesis of VGSCs ligands. This dissertation describes the in vitro biochemical characterization of eleven enzymes involved in PST biosynthesis, including investigating their roles in PST biosynthesis, substrate scope, and catalytic efficiencies. Cyclization, hydroxylation, and sulfation events are critical in the biosynthesis of paralytic shellfish toxins. Enzymes involved in the assembly of the PST tricycle are discussed in Chapters 2 and 3. Many of these enzymes exhibited substrate promiscuity, suggesting they have broad potential as biocatalysts. These studies represent significant contributions to the presently unresolved oxidative cyclization steps in PST biosynthesis. Rieske oxygenases that perform selective C–H hydroxylation reactions on the PST scaffold are the focus of Chapters 4 and 5.

Approaches to reconstituting catalytic activity, assessing substrate preference, and investigating structural features contributing to reaction selectivity are discussed. The selective chemistry performed by these enzymes is unparalleled in comparison to traditional chemical methods, rendering them promising biocatalytic tools. The detoxification of paralytic shellfish toxins by sulfotransferases is discussed in Chapter 6. Sulfate groups decorating the PST scaffold were found to dramatically decrease VGSC binding affinity, and consequently, toxicity. The substrate scope and roles of sulfotransferases in PST biosynthesis are investigated through a lens of biocatalytic utility. Taken together, this research has the potential to provide the tools necessary to enable efficient biocatalytic methods for the synthesis and derivatization of the saxitoxin scaffold, which can be applied to the design of drugs targeting VGSCs for non-opioid pain management and the treatment of neurological disorders. Furthermore, this work is a significant advance toward understanding the underlying chemistry of PST biosynthesis.