Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.
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.
Eiamphungporn
Warawan
W
Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
Soonsanga
Sumarin
S
Lee
Jin-Won
JW
Helmann
John D
JD
D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001555:Benzene Derivatives / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D008041:Linoleic Acids / Q000494:pharmacology; D008054:Lipid Peroxides / Q000494:pharmacology; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D001555:Benzene Derivatives; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016877:Oxidants; D010545:Peroxides; D012333:RNA, Messenger; D012097:Repressor Proteins; C122138:peroxide repressor proteins; C008285:linoleic acid hydroperoxide; D003545:Cysteine; C007164:cumene hydroperoxide