Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

Holger Bauer, Stephan Gromer, Andrea Urbani, Martina Schnölzer, R Heiner Schirmer, Hans-Michael Müller
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引用次数: 53

Abstract

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.

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冈比亚疟蚊的硫氧还蛋白还原酶。
冈比亚按蚊是恶性疟原虫的重要传播媒介。全基因组分析显示,与黑腹果蝇一样,冈比亚果蝇中没有谷胱甘肽还原酶,功能上被硫氧还蛋白系统所取代。该系统的关键酶是硫氧还蛋白还原酶-1,这是一种含有fad的二聚体蛋白,每个亚基55.3 kDa,催化NADPH + H+ +硫氧还蛋白二硫->NADP+ +硫氧还蛋白二硫醇的反应。冈比亚疟蚊trxr基因位于X染色体上,为单拷贝;它代表三个剪接变异体编码两个细胞质变异体和一个线粒体变异体。在大肠杆菌中重组表达了优势亚型冈比亚比亚硫氧还蛋白还原酶-1,并与野生型酶进行了功能比较,最终产量为1.4 U.ml(-1)。在氧化还原滴定中,底物A. gambiae thioredoxin-1 (Km=8.5微米,kcat=15.4 s(-1), pH为7.4,25℃)不能将nadph还原的A. gambiae thioredoxin reducase -1氧化至完全氧化状态。这表明,与其他二硫还原酶不同,冈比亚硫氧还蛋白还原酶-1在催化过程中在四电子还原态和两电子还原态之间振荡。比较了疟疾系统的硫氧还蛋白还原酶。冈比亚拟虫硫氧还蛋白还原酶-1与人类和恶性疟原虫同源物序列同源性分别为52%和45%。这三种酶的主要区别是c端氧化还原中心的结构,反映在催化中间体对自氧化的不同抗性上。该中心的相关序列为冈比亚亚种硫氧还蛋白还原酶的Thr-Cys-Cys-SerOH,人硫氧还蛋白还原酶的gly - cys -硒半胱氨酸- glyoh,恶性疟原虫硫氧还蛋白还原酶的cys - x - x - x - cys - glyoh。这些差异为设计物种特异性抑制剂提供了一种有趣的方法。值得注意的是,冈比亚芽孢杆菌硫氧还蛋白还原酶-1不是一种硒酶,而是含有一个非常不寻常的氧化还原活性Cys-Cys序列。
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