Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues

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• 作者：Mayu Fujikawa ; Kazuo KobayashiYuko Tsutsui ; Takahiro Tanaka ; Takahiro Kozawa
• 刊名：Biochemistry
• 出版年：2017
• 出版时间：January 17, 2017
• 年：2017
• 卷：56
• 期：2
• 页码：403-410
• 全文大小：480K
• ISSN：1520-4995

文摘

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 × 10⁷ M^–1 s^–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.