Division of Environmental Science and Ecological Engineering, Korea University, Anam-Dong 5 Ga 136-713, Seoul, Republic of Korea.
Microbiology (Reading). 2010 May;156(Pt 5):1487-1496. doi: 10.1099/mic.0.034181-0. Epub 2010 Jan 7.
FinR is required for the induction of fpr (ferredoxin-NADP(+) reductase) under superoxide stress conditions in Pseudomonas putida. Many proteobacteria harbour FinR homologues in their genome as a putative LysR-type protein. Three cysteine residues (at positions 150, 239 and 289 in P. putida FinR) are conserved in all FinR homologues. When these conserved cysteines, along with two other cysteine residues present in FinR, were individually mutated to serines, the FinR remained active, unlike SoxR and OxyR in Escherichia coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Among 18 single amino acid mutants, three mutants (T39A, R194A and E225A) abolished fpr induction without any alteration of their DNA-binding ability, whereas other mutants also abrogated their DNA-binding abilities. Interestingly, two mutants (L215P and D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR.
FinR 是 Pseudomonas putida 在超氧胁迫条件下诱导 fpr（ferredoxin-NADP(+) reductase）所必需的。许多 Proteobacteria 在其基因组中都拥有 FinR 同源物，作为一种推定的 LysR 型蛋白。三个半胱氨酸残基（在 Pseudomonas putida FinR 中的位置 150、239 和 289）在所有 FinR 同源物中都保守。当这些保守的半胱氨酸与 FinR 中存在的另外两个半胱氨酸残基分别突变为丝氨酸时，FinR 仍然保持活性，这与大肠杆菌中的 SoxR 和 OxyR 不同。我们用细胞提取物进行的体外 DNA 结合测定的结果表明，FinR 直接结合到 fpr 启动子区域。为了确定 FinR 用于感知超氧胁迫的功能域，我们对 FinR 进行了随机和定点诱变。在 18 个单个氨基酸突变体中，三个突变体（T39A、R194A 和 E225A）在没有任何改变其 DNA 结合能力的情况下消除了 fpr 的诱导，而其他突变体也消除了它们的 DNA 结合能力。有趣的是，两个突变体（L215P 和 D51A）似乎是组成型激活的，而不管超氧胁迫条件如何。二价铁的耗竭、三价铁的添加以及 fdxA（铁氧还蛋白）基因的缺失也参与了 fpr 的调控。这些数据表明，FinR 具有用于氧化还原感应的不寻常残基，并且 FinR 的氧化还原感应机制与众所周知的 OxyR 和 SoxR 机制不同。
