Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar;37(4):1174-81.
doi: 10.1093/nar/gkn1052. Epub 2009 Jan 7.

Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro

Warawan Eiamphungporn  1 , Sumarin Soonsanga, Jin-Won Lee, John D Helmann
Affiliations

Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro

Warawan Eiamphungporn et al. Nucleic Acids Res. 2009 Mar.

Abstract

Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Ribbon structure of scOhrR. The protein structure, based on the solved crystal structure (1) is shown in a side view (A) and top view (B) with the DNA-binding helices located at the bottom in (A). The first monomer is shown in green and the second in blue. The active site C15 residues are in yellow and circled. The dotted line represents a linker between the C-terminus of the first monomer and the N-terminus of the second monomer. Note that only residues Met8 through His144 were visible in the crystal structure (out of 147 residues), so the N- and C-termini are not accurately defined in this model. In (B), the active sites from the N- and C-terminal monomers are indicated. The linker contains five amino acids (GGGGS) (5L) or two repeats of this sequence in the 10L variants.
Figure 2.
Figure 2.
Peroxide-responsiveness of scOhrR in vivo. β-Galactosidase and northern analyses of strains expressing scOhrR and containing an ohrA-cat-lacZ fusion. (A) Cells were either untreated (empty bars) treated (filled bars) with 100 μM CHP for 15 min. (B) Cells were either untreated (empty bars) treated (filled bars) with 5 μM LHP for 15 min. Error bars represent the SD (n = 3). (C) Northern blot analysis of the ohrA transcript in cells with and without CHP treatment, as in (A).
Figure 3.
Figure 3.
Correlation between scOhrR inactivation and S-cysteinylation. (A) FA assays monitoring inactivation of WT-WT (black), WT-C15′S (red) and C15S-WT (blue). The reactions contain 50 nM DNA, 300 nM scOhrR and 10 μM Cys. At 5 min (300 s), 6 μM CHP was added (filled arrowhead), and 10 mM DTT was added after completion of inactivation (open arrowhead). (B–D) ESI–MS analysis of scOhrR proteins (as indicated) prior to and 10 and 30 min after CHP addition (times indicated by thin arrows in panel A) in reactions parallel to those in (A), but without DTT addition.
Figure 4.
Figure 4.
Sensitivity of scOhrR to CHP with and without 10 μM cysteine. FA assays monitoring inactivation of WT-WT (black), WT-C15′S (red), C15S-WT (blue) and C15S, G120C-WT (green). The reactions contained 50 nM DNA, 300 nM scOhrR with (A) or without (B) 10 μM Cys. At 5 min, 6 μM CHP was added (closed arrowhead), and 10 mM DTT was added to reactivate OhrR as indicated by open arrowheads.
Figure 5.
Figure 5.
Sensitivity of scOhrR to LHP with and without 1 mM cysteine. FA assays monitoring inactivation of WT-WT (black), WT-C15′S (red) and C15S-WT (blue) and C15S, G120C-WT (green). The reactions contained 50 nM DNA, 300 nM scOhrR with (A) or without (B) 1 mM Cys. At 5 min, 1 μM LHP was added (closed arrowhead), and 10 mM DTT was added to reactivate OhrR as indicated by open arrowheads.
See this image and copyright information in PMC

References

    1. Hong M, Fuangthong M, Helmann JD, Brennan RG. Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family. Mol. Cell. 2005;20:131–141. - PubMed
    1. Mongkolsuk S, Helmann JD. Regulation of inducible peroxide stress responses. Mol. Microbiol. 2002;45:9–15. - PubMed
    1. Fuangthong M, Atichartpongkul S, Mongkolsuk S, Helmann JD. OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis. J. Bacteriol. 2001;183:4134–4141. - PMC - PubMed
    1. Panmanee W, Vattanaviboon P, Eiamphungporn W, Whangsuk W, Sallabhan R, Mongkolsuk S. OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli. Mol. Microbiol. 2002;45:1647–1654. - PubMed
    1. Panmanee W, Vattanaviboon P, Poole LB, Mongkolsuk S. Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor and regulator of organic hydroperoxide stress. J. Bacteriol. 2006;188:1389–1395. - PMC - PubMed

Publication types

MeSH terms