Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues
Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues
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SoxR（[2Fe-2S] 转录因子）中超氧化物敏感性的合理调节：物种特异性赖氨酸残基的影响
DOI：
10.1021/acs.biochem.6b01096
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
and T. Kozawa
中科院分区：
文献类型：
--
作者：
M. Fujikawa;K. Kobayashi;Y. Tsutsui;T. Tanaka;and T. Kozawa
在大肠杆菌中，[2Fe-2S] 转录因子 SoxR 充当氧化应激传感器。 SoxR 中的转录活性受 [2Fe-2S] 簇的可逆氧化和还原调节。我们之前提出超氧化物 (O2•–) 作为 E 信号具有直接作用。 coliSoxR 和 E 的敏感性。大肠杆菌SoxR 对O2•的反应比铜绿假单胞菌SoxR 高10 倍。两个同系物之间的差异反映了 O2•– 激活调节作用的种间差异。为了研究 SoxR 对 O2•– 敏感性的决定因素，我们替换了肠道细菌 SoxR 同系物中不保守的几个氨基酸，并使用脉冲放射分解研究了 SoxR 与 O2•– 的相互作用。 E的替代。 coliSoxR Lys 残基 89 和 92 以及 Ala 残基 (K89AK92A) 位于 [2Fe-2S] 簇附近，极大地影响了该蛋白质与 O2•– 的反应。该反应的二级速率常数为3.3×107M–1s–1，比野生型SoxR小10倍。相反，P 中 Ala90 相应地被 Lys 取代。 aeruginosaSoxR 使速率增加约 10 倍。相反，将Arg127Ser128Asp129 → Leu127Gln128Ala129 取代引入E。 coliSoxR，以及 P 中相应的取代 (Leu125Gln126Ala127 → Arg125Ser126Asp127)。 aeruginosaSoxR，不影响反应速率。此外，E中的Lys突变。通过测量百草枯响应的 β-半乳糖苷酶表达，coliSoxR (K89AK92A) 显示体内转录活性缺陷。我们的研究结果清楚地支持了这一观点：Lys 对于 O2• 的反应以及 SoxR 的进一步转录活性至关重要。
InEscherichia 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 forE. coliSoxR and that the sensitivity of theE. coliSoxR response to O2•–is 10-fold higher than that ofPseudomonas aeruginosaSoxR. 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 ofE. coliSoxR 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 inP. aeruginosaSoxR increased the rate approximately 10-fold. In contrast, introductions of the Arg127Ser128Asp129 → Leu127Gln128Ala129 substitution intoE. coliSoxR, and the corresponding substitution (Leu125Gln126Ala127 → Arg125Ser126Asp127) inP. aeruginosaSoxR, did not affect the reaction rates. In addition, the Lys mutation inE. coliSoxR (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.