Graduate Thesis Or Dissertation
 

Model systems for structural investigations into peroxiredoxin catalysis, conformation change, and inactivation

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  • Peroxiredoxin (Prx) enzymes catalyze the reduction of hydrogen peroxide, peroxynitrous acid, and organic peroxides, and are extremely efficient peroxidases, with k[subscript cat]/K[subscript M] on the order of 10⁷ - 10⁸ M⁻¹ s⁻¹. Besides their role in oxidative stress defense, evidence has accumulated that some eukaryotes, including humans, use Prxs as switches to regulate peroxide levels for the purpose of signaling events triggered by hormones and growth factors. Their significance and relevance to human health is underscored by the occurrence of cancer in some Prx knockout mice; the overexpression of Prxs in certain cancers, and knockout/knockdown studies that show Prxs in pathogens can be important, and even essential, for pathogen viability and infectivity. Further, their ubiquity in all the kingdoms of life implies Prxs provide indispensible functions. This thesis reports on work aimed at characterizing aspects of Prx function and catalysis using model systems that behave well for experimentation, specifically focusing on detangling the multifaceted roles of conserved residues, catalytic conformation change, and hyperoxidative inactivation. Five chapters of original work include two review articles and three primary research reports. One review provides a relatively broad overview of Prx structure-function (Chapter 2) and the other focuses on observations related to understanding the physiological role(s) of Prxs especially summarizing the results of knockout/knockdown studies and assessing the natural distribution of an enzyme, sulfiredoxin, that is able to reactivate hyperoxidized Prxs (Chapter 3). The latter shows that many virulent bacterial and eukaryotic pathogens lack sulfiredoxin, implying that they are unable to rescue hyperoxidatively inactivated Prxs. Of the primary research reports, two studies using the model Prx Salmonella typhimurium alkyl hydroperoxide reductase C (StAhpC) assess the impact of modifications on structure and dynamics (Chapter 4) and define the roles of highly conserved residues as they pertain to catalysis, conformation change, and oligomerization (Chapter 5). The studies with StAhpC were enabled by the discovery that a previously studied crystal form of the locally-unfolded (LU) conformation of StAhpC is also able to accommodate the fully-folded (FF) conformation. The work includes presentations of the first crystal structure of the wild type enzyme in its substrate-ready form and also the structures of eight mutants of residues that are well-conserved in the Prx1 subfamily of Prxs. The work led to an awareness of how small shifts in the relative stabilities of the FF versus LU conformations could strongly influence Prx function, and this in turn led to the proposal of a novel idea for the design of selective inhibitors of Prxs as potential drug leads: to target regions involved in the catalytic conformation change to trap them in inactive states. The third primary research report (Chapter 6) presents an analysis of three crystal structures of the PrxQ subfamily that had been solved and deposited in the Protein Data Bank by structural genomics groups, but not described in publications. These three structures provided views of the only remaining undescribed type of Prx conformation change - that of the PrxQ group with a resolving Cys in helix 2. In addition to describing the conformation change, we also define roles for conserved residues from a structural perspective for this entire PrxQ subgroup. Finally, in a forward looking part of the thesis (Chapter 7) I describe initial work toward developing Xanthomonas campestris PrxQ (XcPrxQ) as a new model system for study. This enzyme has the advantage of being a monomer, unusual for Prxs, and this makes it more ideal for probing questions related to dynamics. The preliminary work with this system includes crystal structures solved at 1 Å resolution, the highest for any Prx, trapping all relevant active site oxidation states and a ligand-bound form, NMR backbone assignments for the reduced and oxidized forms, and in silico docking analyses aimed at discovering a conformation-stabilizing inhibitor. Furthermore, results showing that the protein in the crystal has strongly enhanced sensitivity to hyperoxidation validates my proposal that inhibitors stabilizing the FF conformation of Prxs will lead to hyperoxidative inactivation. Together, these results establish XcPrxQ as a promising model system for future study.
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