Semisynthesis and characterization of mammalian thioredoxin reductase
Semisynthesis and characterization of mammalian thioredoxin reductase
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DOI：
10.1021/bi0517887
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发表时间：
2006-04-25
期刊：
影响因子：
2.9
通讯作者：
Hondal, RJ
中科院分区：
文献类型：
--
作者：
Eckenroth, B;Harris, K;Hondal, RJ
硫氧还蛋白还原酶和硫氧还蛋白构成了细胞内硫氧还蛋白系统，该系统提供了许多细胞内靶二硫化物的还原等价物。哺乳动物的硫氧还蛋白还原酶含有稀有的氨基酸硒半胱氨酸。硒半胱氨酸被称为“第21个”氨基酸，通过重新编码UGA终止密码子插入到蛋白质中。一些模式真核生物缺乏插入硒半胱氨酸的能力，原核生物具有不同于真核生物的记录装置，从而使哺乳动物硒蛋白的异源表达变得困难。在这里，我们提出了一种半合成的方法来制备哺乳动物硫氧还蛋白还原酶。这种方法在大肠杆菌细胞中产生小鼠硫氧还蛋白还原酶-3的第一个487个氨基酸作为内含素融合蛋白。然后是缺失的含硒半胱氨酸的C末端三肽。通过表达蛋白连接将其连接到硫酯标记的蛋白上。我们以这种方式生产的半合成版本的硫氧还蛋白还原酶对硫氧还蛋白的k(CAT)值在1500-2220min(-1)之间，并且具有很强的过氧化物酶活性，表明该酶是一种功能性形式。以6株L(4 mg/L)为出发菌株，生产了总产率为24 mg的半合成硫氧还蛋白还原酶。这种方法可以生产一种全功能的、半合成的、适合于结构功能研究的硒酶。还报道了第二个半合成系统，该系统利用多肽互补来产生部分活性的酶。我们的多肽互补研究结果表明，不能与酶连接的四肽(Ac-Gly-Cys-Sec-Gly)可以与截短酶形成非共价复合体，形成弱复合体。这种非共价肽-酶复合体的活性比通过肽连接产生的半合成酶低350-500倍。
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.