Watabe S, Makino Y, Ogawa K, Hiroi T, Yamamoto Y, Takahashi S Y
Radioisotope Laboratory, Faculty of Agriculture, Yamaguchi University, Japan.
Eur J Biochem. 1999 Aug;264(1):74-84. doi: 10.1046/j.1432-1327.1999.00578.x.
Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.
线粒体硫氧还蛋白还原酶是从牛肾上腺皮质中纯化得到的。该酶是线粒体硫氧还蛋白依赖性过氧化物还原酶系统中的首个蛋白质组分。纯化后的还原酶在SDS/PAGE上显示出约56 kDa的表观分子量，而天然蛋白约为100 kDa，表明其为同二聚体结构。它催化NADPH依赖性还原5,5'-二硫代双(2-硝基苯甲酸)以及来自不同来源的硫氧还蛋白，但不催化谷胱甘肽、氧化型二硫苏糖醇、DL-α-硫辛酸或胰岛素。氨基酸和核苷酸序列分析表明，它具有由21个氨基酸组成的前导序列，该序列具有线粒体靶向信号的特征。成熟蛋白的氨基酸序列与牛胞质硫氧还蛋白还原酶的序列相似性为57%，与人类谷胱甘肽还原酶的序列相似性为34%，与大肠杆菌(19%)或酵母(17%)的酶序列相似性较低。最近发现人和牛的胞质硫氧还蛋白还原酶含有硒代半胱氨酸(Sec)作为其氨基酸成分之一。我们还通过质谱和C端片段的氨基酸序列分析，在线粒体(mt)-硫氧还蛋白还原酶的C端区域鉴定出了Sec。在所有含Sec的硫氧还蛋白还原酶中保守的四氨基酸基序Gly-Cys-Sec-Gly，可能作为该酶的第三个氧化还原中心发挥作用，因为线粒体还原酶受到1-氯-2,4-二硝基苯的抑制，据报道该物质可共价修饰Sec和Cys。已知哺乳动物硫氧还蛋白还原酶在亚基分子量和结构域结构等方面与细菌或酵母的酶不同。这两种具有相似活性的不同类型的酶被认为是趋同进化而来的。我们的数据清楚地表明，可能起源于共生原核生物的线粒体，含有与胞质酶相似且与细菌酶不同的硫氧还蛋白还原酶。
