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Nucleic Acids Research Advance Access originally published online on January 7, 2009
Nucleic Acids Research 2009 37(4):1174-1181; doi:10.1093/nar/gkn1052
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Nucleic Acids Research, 2009, Vol. 37, No. 4 1174-1181
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

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

Warawan Eiamphungporn1, Sumarin Soonsanga1, Jin-Won Lee2 and John D. Helmann1,*

1Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA and 2Department of Life Science, Hanyang University, Seoul, South Korea

*To whom correspondence should be addressed. Tel: +1 607 255 6570; Fax: +1 607 255 3904; Email: jdh9{at}cornell.edu

Received October 31, 2008. Revised December 16, 2008. Accepted December 17, 2008.

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.

Present address: Sumarin Soonsanga, Engineering Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand


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