PLP-Dependent Biocatalysis As Related to Saxitoxin Biosynthesis
Creators: name: Ackenhusen, Sarah
Publisher: name: My University
Year: 2023
Subjects: subject: biocatalysis
subject: PLP-dependent enzymes
subject: amino acid modification
subject: saxitoxin
subject: acyl carrier protein
subject: Biological Chemistry
subject: Science

description: Biocatalysis has greatly expanded recently due to advances in DNA sequencing, enzyme characterization, and directed evolution. Enzymes are sought after catalysts due to their ability to perform their reactions in water, without the use of harsh chemicals, with exquisite site-, chemo-, and stereo-selectivity. Pyridoxal phosphate (PLP)-dependent enzymes are of interest due to their ability to perform transformations on amine-containing small molecules without the use of protecting groups. This thesis focuses on SxtA, the first enzyme in the biosynthetic pathway towards saxitoxin, a neurotoxin produced by cyanobacteria and dinoflagellates that is found in contaminated shellfish. It addresses both the final domain SxtA α-oxoamine synthase (AOS), a PLP-dependent enzyme that natively converts L-Arg into L-Arg ethyl ketone, and the third domain the acyl carrier protein (ACP). The first chapter provides an overview of enzymes that modify amino acids, with a focus on PLP-dependent enzymes. Chapter 2 focuses on substrate binding of acyl-thioesters in SxtA AOS to overcome the use of expensive acyl-CoA substrates in biosynthesis involving CoA and ACP domains. To address this challenge, this chapter investigated the mechanism of coenzyme A in gating catalysis. SxtA AOS and variants were investigated against a panel of substrates, determining that activity was modulated through the binding of a phosphopantetheine arm to residue Lys154. In situ transthioesterification was also found to be an economical solution to productive reactivity, specifically between pantetheine and simple thioester substrate precursors, improving conversion almost one hundred-fold compared to propionyl-SNAC alone. These findings outline a strategy for employing ACP- and CoA-dependent enzymes that are inaccessible through other means without the need for cost-prohibitive coenzyme A or carrier protein-activated substrates. Furthering the use of SxtA AOS as a biocatalyst, Chapter 3 details the evolution of SxtA AOS to perform aldol reactions with pentanal and its native substrate L-Arg. In this chapter, 32 mutations were incorporated over seven rounds of mutagenesis, achieving an overall 1200-fold improvement in activity with native substrate L-Arg and aldehyde pentanal. Early variants focused on overall stabilization of the enzyme, while later variants improved overall yield and low catalyst loading. Final variants achieved a 11% conversion to the aldol product. Chapter 4 switches focus to SxtA ACP, and the identification of the acyltransferase likely to load SxtA ACP with a malonyl-group from malonyl-CoA. In this chapter, two hypotheses are considered: first, that SxtR, a conserved protein in the saxitoxin gene cluster, might achieve this transformation, and second, that another protein in the organism but outside the gene cluster might perform the acyltransfer. Through careful purification of SxtR from native E. coli protein FabD, this chapter showed SxtR is unlikely to perform the acyltransfer. Instead, it is likely that enzymes similar to E. coli FabD is responsible for the initial acyltransfer. Finally, Chapter 5 concludes the thesis with a summary of each chapter and future directions of the research contained in this thesis. This thesis looks to expand the use of PLP-dependent enzymes as biocatalysts, in the lens of SxtA AOS. This thesis also aims to understand the interactions between enzymes that use acyl-CoA and acyl-ACP domains, as these substrates are expensive or require stoichiometric protein to use, both prohibitive for use in biocatalysis. We anticipate many of the lessons obtained during this research can be more broadly applied in the study of PLP-dependent biocatalysis, enzyme evolution, and ACP acylation.