Browsing by Subject "acyl carrier protein"

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.

Acyl-carrier-protein (ACP) is the principal transporter of fatty acids, coordinating acyl-transfer among a vast network of diverse enzymes and biochemical processes. ACP association with protein partners is thought to be exceedingly transient. This paradigm has posed challenges for understanding the molecular basis for acyl-delivery and dissociation. During biosynthesis of the lipid A component (endotoxin) of lipopolysaccharides, ACP shuttles acyl-intermediates thioester-linked to its 4'-phosphopantetheine arm among four acyltransferases: LpxA, LpxD, LpxL, and LpxM. LpxA and LpxD are essential cytoplasmic enzymes, which not only provide an excellent model system to study ACP-based interaction, but also offer an important therapeutic target for development of novel antibiotics. The current dissertation reports the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and breakage products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release. Moreover, the crystal structure of E. coli LpxA in complex with one form of ACP (holo-ACP) is presented. The structure reveals three molecules of holo-ACP localize to the C-terminal domain of the LpxA homotrimer, and shows the functional role of this domain is two-fold: ACP recognition and nucleotide binding of UDP-GlcNAc. A comparison with the LpxD:ACP complexes uncovers that ACP utilizes different surface residues for recognition even amongst closely related acyltransferases, yet still relies on "electrostatic steering" for docking to its enzyme partner. Insights gleaned from the presented structures have provided not only a better understanding of ACP interaction with acyltransferases, but also has identified the "drugable molecular landscape" for the development of novel antibiotics against infective bacteria.