Fujikawa Mayu, Kobayashi Kazuo, Tsutsui Yuko, Tanaka Takahiro, Kozawa Takahiro
The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan.
Biochemistry. 2017 Jan 17;56(2):403-410. doi: 10.1021/acs.biochem.6b01096. Epub 2017 Jan 4.
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 (O) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O activation. To investigate the determinants of SoxR's sensitivity to O, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O 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 O. The second-order rate constant of the reaction was 3.3 × 10 M s, 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 O and further transcriptional activity of SoxR.
在大肠杆菌中，[2Fe-2S]转录因子SoxR作为氧化应激的传感器发挥作用。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（O）直接作为大肠杆菌SoxR的信号起作用，并且大肠杆菌SoxR对O的反应敏感性比铜绿假单胞菌SoxR高10倍。这两种同源物之间的差异反映了O激活调节作用的种间差异。为了研究SoxR对O敏感性的决定因素，我们替换了肠道细菌SoxR同源物中几个不保守的氨基酸，并使用脉冲辐射分解研究了SoxR与O的相互作用。将大肠杆菌SoxR靠近[2Fe-2S]簇的赖氨酸残基89和92替换为丙氨酸残基（K89AK92A），极大地影响了该蛋白与O的反应。该反应的二级速率常数为3.3×10 M s，比野生型SoxR小10倍。相反，在铜绿假单胞菌SoxR中用赖氨酸替换相应的丙氨酸90使速率增加了约10倍。相比之下，将大肠杆菌SoxR中的Arg127Ser128Asp129替换为Leu127Gln128Ala129，以及在铜绿假单胞菌SoxR中进行相应的替换（Leu125Gln126Ala127→Arg125Ser126Asp127），并不影响反应速率。此外，通过测量百草枯刺激下β-半乳糖苷酶的表达，大肠杆菌SoxR中的赖氨酸突变体（K89AK92A）在体内转录活性方面表现出缺陷。我们的研究结果清楚地支持了赖氨酸对SoxR对O的反应及进一步转录活性至关重要这一观点。
