title: Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 1. Purification and enzymatic activities of overexpressed AhpF and AhpC proteins.
abstract: The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties. Recombinant proteins were isolated in high yield (25-33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. coli cells. We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein. All assays gave identical results whether overexpressed S. typhimurium proteins from E. coli or proteins isolated directly from S. typhimurium were used. Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system. AhpF possesses multiple pyridine nucleotide-dependent activities [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhydrogenase, and, in the presence of AhpC, peroxide reductase activities]. Although AhpF can use either NADH or NADPH as the electron donor for these activities, NADH is the preferred reductant (Km,app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADPH when analyzed using DTNB reductase assays). Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities. In contrast, thiol modification of reduced AhpF does not affect transhydrogenase or oxidase activities. These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides within each protein.
authors.affiliation: Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
authors.forename: L B
authors.initials: LB
authors.lastname: Poole
authors.forename: H R
authors.initials: HR
authors.lastname: Ellis
mesh_terms: D000595:Amino Acid Sequence; D001555:Benzene Derivatives / Q000494:pharmacology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D010544:Peroxidases; D054464:Peroxiredoxins; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D001555:Benzene Derivatives; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C515938:ahpF protein, E coli; C007164:cumene hydroperoxide