title: Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues.
abstract: In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2•-) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O2•-is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O2•-activation. To investigate the determinants of SoxR's sensitivity to O2•-, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O2•-using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein's reaction with O2•-. The second-order rate constant of the reaction was 3.3 × 107M-1s-1, which was 10 times smaller than that of wild-type SoxR. Conversely, the corresponding substitution of Ala90 with Lys in P. aeruginosa SoxR increased the rate approximately 10-fold. In contrast, introductions of the Arg127Ser128Asp129 → Leu127Gln128Ala129 substitution into E. coli SoxR, and the corresponding substitution (Leu125Gln126Ala127 → Arg125Ser126Asp127) in P. aeruginosa SoxR, did not affect the reaction rates. In addition, the Lys mutation in E. coli SoxR (K89AK92A) showed a defect in vivo transcriptional activity by measuring β-galactosidase expression in response to paraquat. Our findings clearly support the idea Lys is critical to the response to O2•-and further transcriptional activity of SoxR.
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Fujikawa
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
authors.forename: Kazuo
authors.identifier: http://orcid.org/0000-0002-5586-9112
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tsutsui
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kozawa
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D011675:Pulse Radiolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D016877:Oxidants; D011994:Recombinant Proteins; D014157:Transcription Factors; D013481:Superoxides; C070724:SoxR protein, Bacteria; D001616:beta-Galactosidase; D008239:Lysine; D000409:Alanine; D010269:Paraquat