Cloning, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus.
The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prx is located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.
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Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D054464:Peroxiredoxins; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology / Q000235:genetics*; D017186:Titrimetry
D001426:Bacterial Proteins; D005420:Flavoproteins; D011994:Recombinant Proteins; D010544:Peroxidases; D054464:Peroxiredoxins; D009247:NADH, NADPH Oxidoreductases; C414565:PrxR protein, Thermus aquaticus