The bacterial alkyl hydroperoxide reductase system serves to protect against the toxic and mutagenic effects of oxidative stress. AhpC, the cysteine-based peroxidase component, is a member of the ubiquitous """"""""peroxiredoxin"""""""" (Prx) family and reduces H2O2 and organic hydroperoxides through transient generation of a cysteine sulfenic acid on the enzyme and subsequent intersubunit disulfide bond formation. AhpF, the flavin-containing reductase component, is present in most, but not all, bacteria and efficiently transfers electrons from NADH (or NADPH) to AhpC. Mammalian Prxs have been implicated in such diverse processes as cellular proliferation and differentiation, immune responses and cell signaling. While most AhpC/Prx homologues are highly expressed and play an important role in oxidative defense, only the AhpC from Helicobacter pylori (the causative agent of gastric ulcers linked to stomach cancer) is known to be absolutely required for viability of that organism. The first specific aim of the proposal focuses on (1) the conformational states, oligomerization and membrane association thought to change during turnover of Salmonella typhimurium AhpC and mammalian Prx II in the presence of peroxides, and (2) the participation of a putative general base catalyst (Arg119) in peroxide reduction by AhpC. The second specific aim explores the mechanism of electron transfers to and from the N-terminal disulfide center of S. typhimurium AhpF. This center (Cys129- Cys132) is part of a distinct redox domain in AhpF known from our studies to mediate electron transfer from redox centers (FAD and Cys345-Cys348) in the C-terminal portion of the protein to AhpC. Our recent crystallographic analyses of AhpF have demonstrated a unique architecture for the N-terminal domain (NTD) and a poorly- characterized homologue, protein disulfide oxidoreductase (PDO), from a thermophile; both NTD and PDO are composed of two intimately-associated thioredoxin-like folds with a putative active site glutamate from the first half acting as a general acid-base catalyst for chemistry at the Cys-X-X-Cys motif of the second half of the domain. Our crystallographic analyses of AhpF also strongly support the involvement of large domain movements in the catalytic cycle of AhpF. Crystallographic and fluorescence approaches will be used in the third specific aim to define the nature of AhpF-AhpC interactions as well as inter- domain interactions within AhpF during intrasubunit electron transfer. Understanding of catalysis by bacterial AhpF and both bacterial and mammalian AhpC homologues will contribute to our knowledge of oxidative stress defense mechanisms and redox-regulated cell signaling in both pathogens and mammalian hosts. Therapeutic intervention in preventing oxidative damage involved in human degenerative diseases, cancer and aging as well as in combating pathogenic defense systems requires a complete molecular and biological understanding of the alkyl hydroperoxide reductase enzymes from both bacterial and human sources.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050389-11
Application #
6693789
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
1993-12-01
Project End
2005-12-31
Budget Start
2004-01-01
Budget End
2004-12-31
Support Year
11
Fiscal Year
2004
Total Cost
$300,560
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Nelson, Kimberly J; Perkins, Arden; Van Swearingen, Amanda E D et al. (2018) Experimentally Dissecting the Origins of Peroxiredoxin Catalysis. Antioxid Redox Signal 28:521-536
Bolduc, Jesalyn A; Nelson, Kimberly J; Haynes, Alexina C et al. (2018) Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins. J Biol Chem 293:11901-11912
Keyes, Jeremiah D; Parsonage, Derek; Yammani, Rama D et al. (2017) Endogenous, regulatory cysteine sulfenylation of ERK kinases in response to proliferative signals. Free Radic Biol Med 112:534-543
Buchko, Garry W; Perkins, Arden; Parsonage, Derek et al. (2016) Backbone chemical shift assignments for Xanthomonas campestris peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics. Biomol NMR Assign 10:57-61
Perkins, Arden; Parsonage, Derek; Nelson, Kimberly J et al. (2016) Peroxiredoxin Catalysis at Atomic Resolution. Structure 24:1668-1678
Poole, Leslie B; Nelson, Kimberly J (2016) Distribution and Features of the Six Classes of Peroxiredoxins. Mol Cells 39:53-9
Parsonage, Derek; Sheng, Fang; Hirata, Ken et al. (2016) X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action. J Struct Biol 194:180-90
Cunniff, Brian; Newick, Kheng; Nelson, Kimberly J et al. (2015) Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma. PLoS One 10:e0127310
Karplus, P Andrew (2015) A primer on peroxiredoxin biochemistry. Free Radic Biol Med 80:183-90
Perkins, Arden; Nelson, Kimberly J; Parsonage, Derek et al. (2015) Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling. Trends Biochem Sci 40:435-45

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