Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, Greifswald, Germany.
Proteomics. 2010 Sep;10(17):3155-64. doi: 10.1002/pmic.201000230.
The MarR/DUF24-type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox-sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6-Ala mutant that the conserved Cys6 apparently contributes to the DNA-binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox-sensing in response to diamide and 2-methylhydroquinone (MHQ) in vivo. DNA-binding experiments indicate that YodB is regulated by a reversible thiol-modification in response to diamide and MHQ in vitro. Redox-regulation of YodB involves Cys6-Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C-terminal Cys101' or Cys108' residues. This study reveals a thiol-disulfide switch model of redox-regulation for the YodB repressor to sense electrophilic compounds in vivo.
MarR/ DUF24 型阻遏物 YodB 响应醌类化合物和联脒在枯草芽孢杆菌中控制偶氮还原酶 AzoR1、硝基还原酶 YodC 和氧化还原感应调节蛋白 Spx。之前，我们使用 yodBCys6-Ala 突变体表明保守的 Cys6 显然有助于 YodB 在体内的 DNA 结合活性。在这里，我们提供的数据表明，Cys6 突变为丝氨酸导致了一种蛋白质形式，该形式在体内对二脒和 2-甲基氢醌 (MHQ) 的氧化还原感应降低。DNA 结合实验表明，YodB 在体外受到二脒和 MHQ 的可逆巯基修饰调节。YodB 的氧化还原调节涉及二脒和醌类化合物在体外通过 Cys6-Cys101' 分子间二硫键形成对 Cys6 的调节。对角线 Western blot 分析证实了 YodB 中形成了需要保守的 Cys6 以及 C 端 Cys101' 或 Cys108' 残基的亚基间二硫键。这项研究揭示了 YodB 阻遏物体内感应亲电化合物的巯基-二硫键开关模型。
