Fernandes Aristi Potamitou, Fladvad Malin, Berndt Carsten, Andrésen Cecilia, Lillig Christopher Horst, Neubauer Peter, Sunnerhagen Maria, Holmgren Arne, Vlamis-Gardikas Alexios
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
J Biol Chem. 2005 Jul 1;280(26):24544-52. doi: 10.1074/jbc.M500678200. Epub 2005 Apr 15.
Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.
谷氧还蛋白是一类广泛存在的蛋白质，可通过还原型谷胱甘肽（GSH）催化二硫键的还原反应。大肠杆菌有三种谷氧还蛋白（Grx1、Grx2和Grx3），均含有典型的二硫醇活性位点CPYC。我们报道了一种新型单硫醇大肠杆菌谷氧还蛋白的克隆、表达及特性研究，将其命名为谷氧还蛋白4（Grx4）。该蛋白由115个氨基酸组成（12.7 kDa），具有一个单硫醇（CGFS）潜在活性位点，与其他单硫醇谷氧还蛋白尤其是酵母Grx5具有高度的序列同源性。基因敲除技术实验表明，编码Grx4的阅读框是必需的。在含有NADPH和谷胱甘肽还原酶的完整体系中，使用GSH和羟乙基二硫醚进行的标准谷氧还蛋白测定中，Grx4作为GSH-二硫键氧化还原酶没有活性。一个工程改造的CGFC活性位点突变体也未获得活性。还原形式的Grx4含有三个硫醇，用氧化型GSH处理会导致谷胱甘肽化并形成二硫键。值得注意的是，Grx4的这种二硫键是NADPH和大肠杆菌硫氧还蛋白还原酶的直接底物，而混合二硫键则由Grx1还原。还原型Grx4显示出将电子转移到氧化型大肠杆菌Grx1和Grx3的潜力。通过特异性酶联免疫吸附测定确定，Grx4高度丰富（750 - 2000 ng/mg总可溶性蛋白），并且在进入稳定期时很可能受鸟苷3',5'-四磷酸调节。缺铁时Grx4水平显著升高，表明该蛋白具有与铁相关的功能。
