Krnajski Z, Gilberger T W, Walter R D, Muller S
Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany.
J Biol Chem. 2000 Dec 29;275(52):40874-8. doi: 10.1074/jbc.M008443200.
The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.
硫氧还蛋白氧化还原系统由依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的同二聚体黄素蛋白硫氧还蛋白还原酶（TrxR）和12 kDa的蛋白硫氧还蛋白组成。它负责还原蛋白质中的二硫键，如核糖核苷酸还原酶和几种转录因子。此外，硫氧还蛋白参与过氧化氢的解毒作用，并保护细胞免受氧化损伤。存在两类TrxR：高分子量（M(r)）和低分子量蛋白。已得到充分表征的大肠杆菌TrxR代表低分子量蛋白家族的成员，而哺乳动物、秀丽隐杆线虫和恶性疟原虫的蛋白则属于高分子量蛋白家族。这些蛋白的一级结构与谷胱甘肽还原酶和硫辛酰胺脱氢酶的结构非常相似。然而，高分子量TrxR除了其具有氧化还原活性的N端半胱氨酸对之外，在其C端还具有一对半胱氨酸残基或一个硒代硫化物基序。这些残基已被证明对硫氧还蛋白的还原至关重要。在本研究中，我们探讨了恶性疟原虫TrxR的活性位点残基是由一个还是两个亚基提供的问题。差异标记的野生型和PfTrxR突变体在大肠杆菌中共表达，重组蛋白种类通过针对重组蛋白各自标签的亲和色谱法进行纯化。PfTrxR野生型和突变蛋白的共表达导致形成三种不同的蛋白种类：同二聚体PfTrxR野生型蛋白、同二聚体突变蛋白以及由一个PfTrxR野生型亚基和一个PfTrxR突变亚基组成的异二聚体。双突变体PfTrxRC88AC535A与PfTrxR野生型的共表达产生了无活性的异二聚体，这表明PfTrxR具有亚基间活性位点。此外，所呈现的数据可能暗示PfTrxR的两个活性位点之间存在协同相互作用。
