A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase
A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase
复制标题
DOI：
10.1074/jbc.m500678200
复制
发表时间：
2005-07-01
影响因子：
4.8
通讯作者：
Vlamis-Gardikas, A
中科院分区：
文献类型：
--
作者：
Fernandes, AP;Fladvad, M;Vlamis-Gardikas, A
谷氧还蛋白是普遍存在的蛋白质，其通过还原型谷胱甘肽（GSH）催化二硫化物的还原。大肠杆菌有三种谷氧还蛋白（Grx 1，Grx 2和Grx 3），都含有经典的二巯基活性位点CPYC。我们报告了一个新的单硫醇E.大肠杆菌谷氧还蛋白，我们将其命名为谷氧还蛋白4（Grx 4）。该蛋白质由115个氨基酸（12.7 kDa）组成，具有一个单硫醇（CGFS）潜在的活性位点，并显示出与其他单硫醇谷氧还蛋白，特别是酵母Grx 5的高度序列同源性。基因敲除技术的实验表明，编码Grx 4的阅读框架是必不可少的。Grx 4作为GSH-二硫化物氧化还原酶在具有NADPH和谷胱甘肽还原酶的完整系统中具有GSH和羟乙基二硫化物的标准谷氧还蛋白测定中无活性。工程改造的CGFC活性位点突变体也没有获得活性。还原形式的Grx 4含有三个硫醇，用氧化GSH处理导致谷胱甘肽化和形成二硫化物。值得注意的是，Grx 4的这种二硫化物是NADPH和E的直接底物。大肠杆菌硫氧还蛋白还原酶，而混合二硫化物被还原Grx 1。还原的Grx 4显示出将电子转移到氧化的E. coli Grx 1和Grx 3。Grx 4是高度丰富的（750 - 2000 ng/mg总可溶性蛋白质），如通过特异性酶联免疫吸附测定所确定的，并且最有可能在进入稳定期时由鸟苷3 '，5'-四磷酸调节。Grx 4在铁耗竭后高度升高，表明该蛋白质具有铁相关功能。
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.