Biochemical Characterization of an Atypical Polyketide Synthase (PKS) from the Apicomplexan Parasite Toxoplasma gondii

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2026-02-07

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2023

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The phylum Apicomplexa encompasses multiple obligate intracellular parasites that pose significant burdens to human health including the causative agents of malaria, toxoplasmosis, and cryptosporidiosis which infect millions of humans and cause hundreds of thousands of deaths each year. During their complex life cycles, apicomplexan parasites coordinate the function of specific proteins to both evade the host immune system and thrive under stressful conditions. Notably, Toxoplasma gondii has been found to harbour multiple polyketide synthase (PKS) genes by bioinformatic analysis, suggesting they can produce secondary metabolite polyketides. While secondary metabolite biosynthetic gene clusters (BGCs) have been known in Apicomplexa for over two decades, limited studies on these enzymes have been completed to date and there have been no characterized products, leaving a void in our understanding of the role of these enzymes in parasite biology. Therefore, characterization of these proteins may aid in our ability to target these biosynthetic enzymes as sources of potential therapeutic candidates in Apicomplexa.While protists are underexplored for biosynthetic potential, research points to this kingdom as an untapped potential for new chemical space. T. gondii for instance possesses multiple putative PKS biosynthetic gene clusters (BGCs) however there have been no secondary metabolite products elucidated thus far. Therefore, our work explores a T. gondii PKS, TgPKS2, and investigates the architecture, predicted structures, and activity of multiple domains within this synthase. Subsequently, Chapters 2 and 3 describes our initial studies on TgPKS2 including hydrolysis activities of acyltransferase (AT) domains, mutagenesis studies, and a first of its kind self-acylation activity of acyl carrier protein (ACP) domains in a modular type I PKS. Chapter 4 further emphasizes the unique attributes of TgPKS2, delving into a never before characterized chain release mechanism, while Chapter 5 compares TgPKS2 transacylation activity to well-characterized bacterial and fungal systems. Combined, these chapters describe our work to biochemically explore TgPKS2, discover the role it plays within the T. gondii life cycle, and further our work to elucidate the metabolite(s) produced by this synthase. Altogether, this research lays the ground work for exploring other apicomplexan and eukaryotic polyketide synthases and significantly increases our knowledge of the biochemical properties of these unique proteins.

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Keeler, Aaron (2023). Biochemical Characterization of an Atypical Polyketide Synthase (PKS) from the Apicomplexan Parasite Toxoplasma gondii. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/30310.

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