Juillan-Binard Céline, Picciocchi Antoine, Andrieu Jean-Pierre, Dupuy Jerome, Petit-Hartlein Isabelle, Caux-Thang Christelle, Vivès Corinne, Nivière Vincent, Fieschi Franck
From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.
the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.
J Biol Chem. 2017 Feb 10;292(6):2485-2494. doi: 10.1074/jbc.M116.752014. Epub 2016 Dec 27.
MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.
MsrPQ是一种新发现的存在于细菌中的蛋氨酸亚砜还原酶系统，它似乎特别参与被次氯酸氧化的周质蛋白的修复。它涉及两种蛋白质：一种周质蛋白MsrP，以前称为YedY，具有Msr活性；以及MsrQ，一种整合的b型血红素跨膜蛋白，它作为MsrP的特定电子供体。MsrQ以前称为YedZ，主要通过生物信息学表征为含血红素膜蛋白FRD超家族的成员，该超家族包括NADPH氧化酶蛋白（NOX/DUOX）。在这里，我们报告了来自[具体来源未提及]的MsrQ蛋白的详细生化特性。我们优化了MsrQ-GFP融合蛋白的过表达和膜溶解条件，并建立了一种纯化方案以生产纯MsrQ。结合紫外可见光谱、血红素定量和组氨酸残基的定点诱变，我们证明MsrQ能够通过MsrQ和NOX蛋白家族之间保守的组氨酸残基结合两个b型血红素。此外，我们鉴定了与真核NOX酶的脱氢酶结构域相关的黄素还原酶Fre，它是MsrQ血红素部分的一种有效的胞质电子供体。交联实验以及表面等离子体共振表明Fre与MsrQ相互作用形成特定复合物。综上所述，这些数据支持了第一个与真核NOX家族相关且参与周质氧化蛋白还原的原核双组分蛋白系统的鉴定。
