Eckenroth Brian, Harris Katharine, Turanov Anton A, Gladyshev Vadim N, Raines Ronald T, Hondal Robert J
Department of Biochemistry, University of Vermont, 89 Beaumont Avenue, Given Laboratory, Room B413, Burlington, Vermont 05405, USA.
Biochemistry. 2006 Apr 25;45(16):5158-70. doi: 10.1021/bi0517887.
Thioredoxin reductase and thioredoxin constitute the cellular thioredoxin system, which provides reducing equivalents to numerous intracellular target disulfides. Mammalian thioredoxin reductase contains the rare amino acid selenocysteine. Known as the "21st" amino acid, selenocysteine is inserted into proteins by recoding UGA stop codons. Some model eukaryotic organisms lack the ability to insert selenocysteine, and prokaryotes have a recoding apparatus different from that of eukaryotes, thus making heterologous expression of mammalian selenoproteins difficult. Here, we present a semisynthetic method for preparing mammalian thioredoxin reductase. This method produces the first 487 amino acids of mouse thioredoxin reductase-3 as an intein fusion protein in Escherichia coli cells. The missing C-terminal tripeptide containing selenocysteine is then ligated to the thioester-tagged protein by expressed protein ligation. The semisynthetic version of thioredoxin reductase that we produce in this manner has k(cat) values ranging from 1500 to 2220 min(-)(1) toward thioredoxin and has strong peroxidase activity, indicating a functional form of the enzyme. We produced the semisynthetic thioredoxin reductase with a total yield of 24 mg from 6 L of E. coli culture (4 mg/L). This method allows production of a fully functional, semisynthetic selenoenzyme that is amenable to structure-function studies. A second semisynthetic system is also reported that makes use of peptide complementation to produce a partially active enzyme. The results of our peptide complementation studies reveal that a tetrapeptide that cannot ligate to the enzyme (Ac-Gly-Cys-Sec-Gly) can form a noncovalent complex with the truncated enzyme to form a weak complex. This noncovalent peptide-enzyme complex has 350-500-fold lower activity than the semisynthetic enzyme produced by peptide ligation.
硫氧还蛋白还原酶和硫氧还蛋白构成细胞硫氧还蛋白系统，该系统为众多细胞内靶二硫键提供还原当量。哺乳动物硫氧还蛋白还原酶含有稀有氨基酸硒代半胱氨酸。硒代半胱氨酸被称为“第21种”氨基酸，通过对UGA终止密码子进行重新编码而插入蛋白质中。一些模式真核生物缺乏插入硒代半胱氨酸的能力，原核生物具有与真核生物不同的重新编码装置，因此使得哺乳动物硒蛋白的异源表达变得困难。在此，我们提出一种制备哺乳动物硫氧还蛋白还原酶的半合成方法。该方法在大肠杆菌细胞中产生作为内含肽融合蛋白的小鼠硫氧还蛋白还原酶-3的前487个氨基酸。然后，通过表达蛋白连接将缺失的含硒代半胱氨酸的C末端三肽连接到硫酯标记的蛋白上。我们以这种方式产生的半合成硫氧还蛋白还原酶对硫氧还蛋白的k(cat)值范围为1500至2220 min⁻¹，并且具有很强的过氧化物酶活性，表明该酶具有功能形式。我们从6升大肠杆菌培养物中以24毫克的总产率（4毫克/升）产生了半合成硫氧还蛋白还原酶。这种方法允许生产一种适合进行结构-功能研究的全功能半合成硒酶。还报道了第二个半合成系统，该系统利用肽互补来产生部分活性的酶。我们的肽互补研究结果表明，一种不能与该酶连接的四肽（Ac-Gly-Cys-Sec-Gly）可以与截短的酶形成非共价复合物，从而形成弱复合物。这种非共价肽-酶复合物的活性比通过肽连接产生的半合成酶低350-500倍。
