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

Eiamphungporn W, Soonsanga S, Lee JW, Helmann JD - Nucleic Acids Res. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.

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.

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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.
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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.

Mentions: To more closely monitor the correlation between protein oxidation and DNA-binding, we purified each of the scOhrR repressors (Supplementary Figure S1) and monitored DNA-binding using a FA-based assay (Figure 3A). As reported previously for the wild-type dimeric protein, the WT-WT scOhrR was completely inactivated by treatment with 6 μM CHP in buffer containing 10 μM free cysteine (13). As expected, protein inactivation was due to S-cysteinylation since activity was rapidly and quantitatively restored by addition of dithiothreitol (DTT).Figure 3.


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

Eiamphungporn W, Soonsanga S, Lee JW, Helmann JD - Nucleic Acids Res. (2009)

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.
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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.
Mentions: To more closely monitor the correlation between protein oxidation and DNA-binding, we purified each of the scOhrR repressors (Supplementary Figure S1) and monitored DNA-binding using a FA-based assay (Figure 3A). As reported previously for the wild-type dimeric protein, the WT-WT scOhrR was completely inactivated by treatment with 6 μM CHP in buffer containing 10 μM free cysteine (13). As expected, protein inactivation was due to S-cysteinylation since activity was rapidly and quantitatively restored by addition of dithiothreitol (DTT).Figure 3.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.

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.

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