Cloning, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (Pr xR) 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 peroxired oxins from a range of organisms and that PrxR resembles NADH-dependent flav oenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacter ia. The sequence of PrxR also resembles those of thioredoxin reductases (Tr xR) from thermophiles but with an N-terminal extension of about 200 residue s. 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 mol ecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respec tively; both enzymes exist as multimers. The recombinant flavoenzyme requir es 3 mol equivalents of dithionite for full reduction, as is consistent wit h 1 FAD and 2 disulfides per monomer, PrxR and Prx together catalyze the an aerobic reduction of hydrogen peroxide. The activity of Prx is much less th an has been observed with homologous proteins. Prx appears to be inactivate d by cumene hydroperoxide. PrxR itself has low peroxidase activity.