Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

The relatively high level of palmitic acid (22 mol%) in cottonseeds may be due in part to the activity of a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE). In embryo extracts, PATE activity was highest at the maximum rate of reserve accumulation (oil and protein). The cotton FatB mRNA transcript abundance also peaked during this developmental stage, paralleling the profiles of PATE enzyme activity and seed oil accumulation. A cotton FatB cDNA clone was isolated by screening a cDNA library with a heterologous Arabidopsis FatB probe (Pirtle et al., 1999, Plant and Cell Physiology 40: 155-163). The predicted amino acid sequence of the cotton PATE preprotein had 63% identity to the Arabidopsis FatB thioesterase sequence, suggesting that the cotton cDNA clone probably encoded a FatB-type thioesterase. When acyl-CoA synthetase-minus E. coli mutants expressed the cotton cDNA, an increase in 16:0 free fatty acid content was measured in the culture medium. In addition, acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a preference for palmitoyl-ACP over oleoyl-ACP in vitro, indicating that the cotton putative FatB cDNA encoded a functional thioesterase with a preference for saturated acyl-ACPs over unsaturated acyl-ACPs (FatA). Overexpression of the FatB cDNA in transgenic cotton resulted in elevated levels of palmitic acid in transgenic somatic embryos compared to control embryos. Expression of the anti-sense FatB cDNA in transgenic cotton plants produced some plants with a dwarf phenotype. These plants had significantly smaller mature leaves, all with smaller cells, suggesting that these plants may have less palmitic acid available for incorporation into extraplastidial membrane lipids during cell expansion. Thus manipulation of FatB expression in cotton directly influenced palmitic acid levels. Collectively, data presented in this dissertation support the hypothesis that there indeed is a palmitoyl-ACP thioesterase in cotton, encoded by the isolated FatB cDNA, which plays a major role in regulating palmitic acid content of extraplastidial complex glycerolipids. This work forms the basis for future studies of the influence of palmitic acid content on plant membrane function and provides a key target for the metabolic engineering of palmitic acid levels in storage oils of developing cottonseeds.

Polyketide secondary metabolites constitute a structurally-diverse and clinically-important family of natural products. The wide range of biological activities represented by these substrates have contributed to therapeutic agents with annual sales exceeding $20B USD. Large multi-domain proteins called polyketide synthases (PKSs) use simple building blocks to generate highly-oxygenated and stereochemically-rich frameworks with astonishing selectivity. These substrates often feature rigidifying biases imposed by macrocyclic lactones and substituted heterocycles, which can impact their bioactive conformation. The work of this dissertation combines synthetic chemistry and biochemistry to investigate chemoenzymatic production of macrocyclic polyketide natural products. Research focused on validating a transannular oxa-conjugate addition strategy to assembly 2,6-cis-tetrahydropyran (THP) ring systems, as demonstrated by synthesis of the macrocyclic core to neopeltolide. Ultimately, we wish to apply this chemistry to de novo PKS pathways for rapid, reliable, and sustainable production of THP-bearing products like neopeltolide, and toward building SAR libraries. Additionally, a second study probed the specificity of the macrolactonizing thioesterase (TE) domain from the 6-deoxyerythronolide B (DEBS) biosynthetic pathway. This pathway is the paradigm for type-I PKS systems, and is responsible for producing the macrolide core of erythromycin. Our on-going research evaluates the limits of promiscuity within this specific catalytic domain, to characterize the structural elements required to accurately predict macrolactonization. The long-term goal of this study is to assess the potential applicability of DEBS TE as a generalized cyclization biocatalyst for combinatorial biochemistry and chemoenzymatic research.

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<div>Cyclic peptides are important sources of medicines. </div><div>They are advantageous compared to linear peptides because they possess lower flexibility, which allows for high-affinity target binding and enhanced proteolytic stability. Unfortunately, achieving head-to-tail cyclization of peptides is quite challenging, as it is hard to control efficiency and regiospecificity of peptide macrocyclization. Many have attempted to improve peptide cyclization, including the use of different synthetic reagents as well as synthetic techniques to allow amide-bond formation and promote cyclization. While these strategies have offered great potential solutions, the aim of this study is to explore an alternative strategy that utilizes biocatalysis as a method of achieving successful peptide cyclization. Biocatalysis is the use of enzymes as natural process catalysts under artificial in vitro conditions. Biocatalysis is often more environmentally friendly and safer compared to traditional organic synthesis methods. Non-ribosomal peptide synthetases (NRPSs) are one of the major sources of cyclic peptides in nature. These are systems of large multifunctional proteins are organized into functional domains that act as an assembly line to generate peptide natural products. Normally, the thioesterase domain is responsible for hydrolysis and cyclization of the peptide. Recently, a novel cyclase (SurE) that is physically discrete from the NRPS was discovered. Based on this unique quality, we hypothesized that SurE would be easier to express compared to thioesterase domains and, for this reason, SurE could be a fantastic biocatalyst for the cyclization of peptides. To test this, we designed and generated an expression vector for SurE. We then expressed and purified the SurE protein. We also synthesized three linear peptides of varying lengths. To test for SurE activity, we attempted to add N-acetylcysteamine (SNAC) to mimic its native substrate. Unfortunately, we were unable to successfully attach the SNAC to our linear peptide. To combat this issue, a new synthesis strategy is currently being developed. This work is currently ongoing in the Parkinson lab, with the aim being to test the SurE protein, as well as other PBP-like cyclases, on other modified linear peptides and demonstrate whether the protein has the ability to cyclase a wide scope of peptides.</div><div><br></div>

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation.