title: The hydrogen peroxide hypersensitivity of OxyR2 inVibrio vulnificusdepends on conformational constraints.
abstract: Most Gram-negative bacteria respond to excessive levels of H2O2using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality.Vibrio vulnificushas two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H2O2sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H2O2was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H2O2and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H2O2sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and.
authors.forename: Inseong
authors.identifier: 0000-0002-7964-4751
authors.initials: I
authors.lastname: Jo
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and.|the National Research Laboratory of Molecular Microbiology and Toxicology, Seoul National University, Seoul 08826, Korea.
authors.forename: Dukyun
authors.initials: D
authors.lastname: Kim
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and.|the National Research Laboratory of Molecular Microbiology and Toxicology, Seoul National University, Seoul 08826, Korea.
authors.forename: Ye-Ji
authors.initials: YJ
authors.lastname: Bang
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and.
authors.forename: Jinsook
authors.initials: J
authors.lastname: Ahn
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and choish@snu.ac.kr.|the National Research Laboratory of Molecular Microbiology and Toxicology, Seoul National University, Seoul 08826, Korea.
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Choi
authors.affiliation: From the Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Research Institute for Agriculture and Life Sciences, and hanc210@snu.ac.kr.
authors.forename: Nam-Chul
authors.identifier: 0000-0003-4813-748X
authors.initials: NC
authors.lastname: Ha
mesh_terms: D000975:Antioxidants / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D014644:Genetic Variation; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055503:Protein Multimerization; D012313:RNA / Q000032:analysis; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D041261:Vibrio vulnificus / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007093:Imidazoles; D012097:Repressor Proteins; D014157:Transcription Factors; D005973:Glutamine; D006639:Histidine; D012313:RNA; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D008239:Lysine; D005998:Glycine
keywords: OxyR; Vibrio vulnificus; antioxidant; bacterial transcription; crystal structure; crystallography; hydrogen peroxide; reactive oxygen species (ROS); sensitivity