The alkyl hydroperoxide reductase (AhpR) enzyme system of Salmonella typhimurium serves to protect these organisms from the toxic and mutagenic effects of oxidative stress. The broad goal of the proposed studies is to determine the structural and functional bases for catalysis by the two proteins, AhpF and AhpC, which comprise this peroxidase system. AhpF, a flavoprotein disulfide reductase, has thus far been identified only in bacteria and could become a useful drug target as new classes of antibacterial agents are sought. In contrast, AhpC homologues have been identified in organisms from every kingdom, suggesting a crucial biological function for this ubiquitous antioxidant protein. The first two specific aims focus on the identification of catalytically-relevant redox centers in AhpF and the elucidation of the interactions which take place between these centers and the active site of AhpC during catalysis. Based on previous studies of truncated and mutant AhpF proteins, the hypothesis to be tested is that the N-terminal disulfide center of AhpF (Cys129-Cys132) is directly involved in electron transfer to AhpC, mediating electron transfer from the thioredoxin reductase-like disulfide redox center (Cys345-Cys348) of AhpF. The necessity for alternating interactions between multiple redox centers in the AhpF/AhpC system has also led to the proposal that conformational changes occur within AhpF during catalytic electron transfer. The third specific aim addresses the putative physiological role for these proteins in reducing lipid hydroperoxides formed through oxidative damage. These studies will assess the protection afforded by the AhpR system against the increased permeability observed on oxidative damage of membranes using model systems. Protection by the peroxidatic activity of AhpC may be critical to the promotion of infection and invasion by S. typhimurium as well as by a number of other human pathogens demonstrated to express homologues of AhpC (e.g. Entamoeba histolytica, the causative agent of amoebic dysentery, a worldwide health problem, and Helicobacter pylori, a common human pathogen causing stomach ulcers and cancer). Antioxidant functions of AhpC homologues may also protect humans against the multitude of health problems caused by oxidative stress.
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