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Title
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Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor and regulator of organic hydroperoxide stress.
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Authors
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W.Panmanee,
P.Vattanaviboon,
L.B.Poole,
S.Mongkolsuk.
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Ref.
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J Bacteriol, 2006,
188,
1389-1395.
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PubMed id
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Abstract
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Xanthomonas campestris pv. phaseoli OhrR belongs to a major family of
multiple-cysteine-containing bacterial organic hydroperoxide sensors and
transcription repressors. Site-directed mutagenesis and subsequent in vivo
functional analyses revealed that changing any cysteine residue to serine did
not alter the ability of OhrR to bind to the P1 ohrR-ohr promoter but
drastically affected the organic hydroperoxide-sensing and response mechanisms
of the protein. Xanthomonas OhrR requires two cysteine residues, C22 and C127,
to sense and respond to organic hydroperoxides. Analysis of the free thiol
groups in wild-type and mutant OhrRs under reducing and oxidizing conditions
indicates that C22 is the organic hydroperoxide-sensing residue. Exposure to
organic hydroperoxides led to the formation of an unstable OhrR-C22 sulfenic
acid intermediate that could be trapped by
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and detected by UV-visible spectral
analysis in an oxidized C127S-C131S mutant OhrR. In wild-type OhrR, the cysteine
sulfenic acid intermediate rapidly reacts with the thiol group of C127, forming
a disulfide bond. The high-performance liquid chromatography-mass spectrometry
analysis of tryptic fragments of alkylated, oxidized OhrR and nonreducing
polyacrylamide gel electrophoresis analyses confirmed the formation of
reversible intersubunit disulfide bonds between C22 and C127. Oxidation of OhrR
led to cross-linking of two OhrR monomers, resulting in the inactivation of its
repressor function. Evidence presented here provides insight into a new organic
hydroperoxide-sensing and response mechanism for OhrRs of the multiple-cysteine
family, the primary bacterial transcription regulator of the organic
hydroperoxide stress response.
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