Limits...
Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Show MeSH

Related in: MedlinePlus

The OhrA peroxiredoxin and the nitroreductase HypO protect cells against hypochlorite toxicity. Growth phenotype of B. subtilis wild-type (WT), ΔohrA and ΔohrAΔhypO mutant strains that were treated with 75 µM NaOCl at an OD500 of 0.4. The growth curves are representives of at least three independent growth experiments. Two biological replicates are shown in Supplementary Figure S3A–S3C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351151&req=5

gkr1316-F2: The OhrA peroxiredoxin and the nitroreductase HypO protect cells against hypochlorite toxicity. Growth phenotype of B. subtilis wild-type (WT), ΔohrA and ΔohrAΔhypO mutant strains that were treated with 75 µM NaOCl at an OD500 of 0.4. The growth curves are representives of at least three independent growth experiments. Two biological replicates are shown in Supplementary Figure S3A–S3C.

Mentions: Previous phenotype analyses have shown that the OhrR-controlled OhrA peroxiredoxin is a specific determinant of NaOCl detoxification (17). Recently, it was shown that HypO shows nitroreductase activity in vitro (47). We analysed the growth phenotype of ΔhypR and ΔhypO single and ΔhypOΔohrA double mutants to investigate whether the HypO nitroreductase contributes to NaOCl resistance. No differences in the sensitivities of the ΔhypO and ΔhypR single mutants were detected compared to the wild-type (Supplementary Figure S3A and S3B). However, the growth of the ΔhypOΔohrA double mutant was significantly more impaired than that of the ΔohrA single mutant after treatment with 75 µM NaOCl (Figure 2 and Supplementary Figure S3C). This indicates that HypO also provides protection against NaOCl stress in B. subtilis.Figure 2.


Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

The OhrA peroxiredoxin and the nitroreductase HypO protect cells against hypochlorite toxicity. Growth phenotype of B. subtilis wild-type (WT), ΔohrA and ΔohrAΔhypO mutant strains that were treated with 75 µM NaOCl at an OD500 of 0.4. The growth curves are representives of at least three independent growth experiments. Two biological replicates are shown in Supplementary Figure S3A–S3C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351151&req=5

gkr1316-F2: The OhrA peroxiredoxin and the nitroreductase HypO protect cells against hypochlorite toxicity. Growth phenotype of B. subtilis wild-type (WT), ΔohrA and ΔohrAΔhypO mutant strains that were treated with 75 µM NaOCl at an OD500 of 0.4. The growth curves are representives of at least three independent growth experiments. Two biological replicates are shown in Supplementary Figure S3A–S3C.
Mentions: Previous phenotype analyses have shown that the OhrR-controlled OhrA peroxiredoxin is a specific determinant of NaOCl detoxification (17). Recently, it was shown that HypO shows nitroreductase activity in vitro (47). We analysed the growth phenotype of ΔhypR and ΔhypO single and ΔhypOΔohrA double mutants to investigate whether the HypO nitroreductase contributes to NaOCl resistance. No differences in the sensitivities of the ΔhypO and ΔhypR single mutants were detected compared to the wild-type (Supplementary Figure S3A and S3B). However, the growth of the ΔhypOΔohrA double mutant was significantly more impaired than that of the ΔohrA single mutant after treatment with 75 µM NaOCl (Figure 2 and Supplementary Figure S3C). This indicates that HypO also provides protection against NaOCl stress in B. subtilis.Figure 2.

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Show MeSH
Related in: MedlinePlus