Tetaud E, Fairlamb A H
Department of Biochemistry, University of Dundee, Scotland, UK.
Mol Biochem Parasitol. 1998 Oct 30;96(1-2):111-23. doi: 10.1016/s0166-6851(98)00120-0.
As a consequence of aerobic metabolism, trypanosomatids are exposed to reactive oxygen intermediates such as superoxide, hydrogen peroxide and the hydroxyl radical. Metabolism of hydrogen peroxide in Crithidia fasciculata is accomplished by three distinct proteins, tryparedoxin, tryparedoxin peroxidase and trypanothione reductase, working in concert with the substrates NADPH and trypanothione. Here, we report the cloning and characterisation of the tryparedoxin (TryX) and tryparedoxin peroxidase (TryP) genes from C. fasciculata. Both genes are multicopy and organized in distinct tandem arrays in the genome. TryX encodes a 16 kDa protein, which belongs to the thioredoxin superfamily, sharing the WCPPC motif, whereas TryP encodes a 21 kDa protein belonging to a new class of peroxidases called 2-Cys peroxidoxins. Both TryX and TryP were expressed in Escherichia coli and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione and trypanothione reductase, similar to the native proteins. TryX is rapidly reduced by trypanothione, but weakly by glutathionylspermidine, glutathione or ovothiol A. TryP shows a broad substrate specificity and can reduced hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide with equal efficiency.
由于有氧代谢，锥虫会接触到超氧化物、过氧化氢和羟基自由基等活性氧中间体。在 fasciculata 克氏锥虫中，过氧化氢的代谢是由三种不同的蛋白质——锥虫硫氧还蛋白、锥虫硫氧还蛋白过氧化物酶和锥虫硫醇还原酶，与底物 NADPH 和锥虫硫醇协同作用来完成的。在此，我们报告了来自 fasciculata 克氏锥虫的锥虫硫氧还蛋白（TryX）和锥虫硫氧还蛋白过氧化物酶（TryP）基因的克隆和特性。这两个基因都是多拷贝的，并且在基因组中以不同的串联阵列形式排列。TryX 编码一种 16 kDa 的蛋白质，属于硫氧还蛋白超家族，具有 WCPPC 基序，而 TryP 编码一种 21 kDa 的蛋白质，属于一类名为 2 - 半胱氨酸过氧化物酶的新的过氧化物酶。TryX 和 TryP 都在大肠杆菌中表达，纯化后的重组蛋白在 NADPH、锥虫硫醇和锥虫硫醇还原酶存在的情况下能够利用过氧化氢，这与天然蛋白相似。TryX 能被锥虫硫醇迅速还原，但被谷胱甘肽亚精胺、谷胱甘肽或卵硫醇 A 还原的能力较弱。TryP 表现出广泛的底物特异性，能够以相同效率还原过氧化氢、叔丁基过氧化氢和异丙苯过氧化氢。
