In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli.
The OxyR protein is a transcriptional activator for a subset of peroxide stress-inducible genes, most of which are involved in defense systems against oxidative stress. Recently, it was demonstrated that purified OxyR has one intramolecular disulfide bond, which led to the proposal that the reversible disulfide bond formation regulates the activity of OxyR as a transcription factor in response to peroxide stress. In this study, I demonstrated by SDS-PAGE under non-reducing conditions that an intramolecular disulfide bond is formed in OxyR upon exposure of the cells to hydrogen peroxide in vivo. Experiments using strains expressing mutant OxyR proteins with Cys to Ser single amino acids substitutions confirmed that the disulfide bond is formed between the Cys-199 and -208. Kinetic analyses indicated that the formation of the disulfide bond is rapid and transient, oxidized within 30 s and re-reduced within 5 min after the addition of hydrogen peroxide in the wild-type strain. These results provide evidence for the regulatory role of the reversible oxidation of dithiol to disulfide in sensing peroxide stress in vivo and signal transduction to the transcription apparatus by OxyR.
Tao
K
K
Radioisotope Center, University of Tokyo, Japan. tao@ric.u-tokyo.ac.jp
D003545:Cysteine / Q000378:metabolism; D004268:DNA-Binding Proteins; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014169:Transformation, Bacterial
D004268:DNA-Binding Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C499299:oxyR protein, E coli; D012694:Serine; D006861:Hydrogen Peroxide; D003545:Cysteine