Novel functions of peroxiredoxin Q from Deinococcus radiodurans R1 as a peroxidase and a molecular chaperone

Abstract

Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double-deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild-type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans.

Keywords: DR0846; Deinococcus radiodurans R1; molecular chaperone; peroxidase; peroxiredoxin Q.

Figure 1

Expression analysis of Prx genes in response to oxidative stress or gamma rays. Cells were grown on TGY medium for 16 h at 30 °C. mRNA levels of peroxiredoxin genes were determined by qRT‐PCR after treatment with 20 mm H₂O₂ for 5–30 min (A) or treatment with gamma rays at 1–5 kGy (B). Data are means ± SE from three replications for each treatment. Different letters indicate significant differences at P < 0.05 between the groups by one‐way ANOVA with Tukey's test. DR1343 (gap) was used as a loading control. DR1998 (KatE1) and DR2340 (recA) were used as positive controls for oxidative stress and gamma rays, respectively.

Figure 2

Purity and oligomeric state of DR0846 based on SDS/PAGE (A), native‐PAGE (B), and SEC (C). The proteins were denatured by heating in the presence (R) or absence (N) of 5 mm DTT. DR0846 was separated by 12% SDS/PAGE (A) or 10% native‐PAGE (B) and the gel was stained with Coomassie Blue. M, Marker; R, Reducing; N, Nonreducing. SEC was performed using a Superdex 200 10/300 column. The numbers in the chromatogram represent the molecular weights of the standard proteins; blue dextran (> 2000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), ovalbumin (44 kDa), and carbonic anhydrase (29 kDa).

Figure 3

Peroxidase and chaperone activities of DR0846 and DR0846 Cys mutant proteins. (A) Peroxidase enzyme assay of DR0846. Peroxidase enzyme activity was measured using the yeast Trx system at various concentrations. (B) Molecular chaperone assay of DR0846. Chaperone activity was measured by the aggregation of MDH at 42 °C at different molar ratios of MDH/DR0846. Con., 1 : 0 (●); 1 : 0.5 DR0846 (○); 1 : 1 DR0846 (♦); 1 : 2 DR0846 (♢). Peroxidase enzyme assay (C) and chaperone enzyme assay (D) of DR0846 Cys mutant. C60S, C65S, and C60S/C65S are cysteine substitution mutants. Data are means ± SD of three independent experiments. Different letters indicate significant differences at P < 0.05 between the WT and mutant proteins by one‐way ANOVA with Tukey's test.

Figure 4

Redox properties of DR0846 and Cys mutant proteins. AMS shift assays were performed using purified WT DR0846 and cysteine mutant proteins. Purified proteins were precipitated with TCA, and treated without (−) or with (+) AMS. The proteins were resolved by reducing SDS/PAGE and subjected to a western blot analysis. Asterisks indicate the oxidized forms of each protein.

Figure 5

Hydrogen peroxide (H₂O₂) and heat stress tolerance assays of the ∆dr0846 mutant. Exponential‐phase WT Deinococcus radiodurans and the deletion mutant (Δdr0846) were grown on TGY plates containing different concentrations of H₂O₂ for 16 h (A) or incubated at 30 °C or 42 °C for 30 min and grown on TGY plates for 16 h, followed by serial dilution (B).