Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor

Abstract

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.

Analyses of the Regulatory Mechanism and Physiological Roles of Pseudomonas aeruginosa OhrR, a Transcription Regulator and a Sensor of Organic Hydroperoxides

“…6); however, addition of 1 mM DTT could not reestablish OhrRC121S DNA binding activity. The results are consistent with the recent hypothesis that the conserved cysteine at the C terminus plays a role in prevention of the overoxidation of the redox-sensing cysteine (33,34). The overoxidized forms of cysteine are sulfinic acid and sulfonic acid, which cannot be reduced by DTT (17).…”

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

“…MarR homologues control pathways that are critical to bacterial physiology such as stress responses (41,42), virulence (43), metabolism (10), and multiple-drug resistance (23,44,45). The activity of a subset of these proteins can be modified through the formation of a disulfide bond (19,41,46). However, most respond to specific ligands.…”

Crystal Structures of the Reduced, Sulfenic Acid, and Mixed Disulfide Forms of SarZ, a Redox Active Global Regulator in Staphylococcus aureus

“…Although helix ␣5 in SarZ-BT does not reorganize to the extent of helix ␣5 in XcOhrR upon modification, it does shorten and bend, suggesting a conserved area of flexibility in the MarR family. Further, a recent study on BsOhrR converted the one-cysteine protein to a two-cysteine protein by introducing a second cysteine in a spot analogous to that of the second cysteine in XcOhrR (48). When exposed to oxidants, the mutated BsOhrR formed a disulfide bond.…”