Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.
Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.
复制标题
刚果锥虫的锥硫酮还原酶：基因分离、一级序列测定以及与谷胱甘肽还原酶的比较。
DOI：
10.1021/bi00414a010
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Walsh,CT
中科院分区：
文献类型：
--
作者：
Shames,SL;Kimmel,BE;Peoples,OP;Agabian,N;Walsh,CT
修订稿于 1988 年 1 月 22 日收到摘要：编码锥硫酮还原酶的基因，即寄生锥虫特有的含氧化还原二硫化物的黄素酶（Shames 等，1986），已从牛病原体刚果锥虫中分离出来。使用含有肌苷的寡核苷酸探针编码序列进行文库筛选，所述探针编码从纯化的束状短膜霉酶中分离的两个活性位点肽确定的序列。根据Sanger双脱氧链终止法测定该基因的核苷酸序列。结构基因长1476个核苷酸，编码492个氨基酸。我们已经鉴定出含有氧化还原活性二硫键的活性位点肽，该肽对应于人红细胞谷胱甘肽还原酶的组氨酸-467区域，以及在所有含有二硫键的黄素蛋白还原酶中高度保守的黄素结合域。从 C. fasciculata 锥硫酮还原酶中分离出的 5 个胰蛋白酶肽（80 个残基）与 T. congolense 酶的一级序列的比对显示出 88% 同源性和 76% 同一性。另外，来自大肠杆菌或人红细胞的谷胱甘肽还原酶与刚果锥虫锥硫酮还原酶的序列比较揭示了> 50%的同源性。对在人红细胞谷胱甘肽还原酶的结合/催化中具有功能活性的锥硫酮还原酶的一级序列中的氨基酸残基的搜索表明，只有两个精氨酸残基（Arg-37和Arg-347）不存在，X射线晶体学数据表明与GSt谷胱甘肽甘氨酰羧酸盐形成氢键。锥虫寄生虫是人类和家畜多种严重疾病的病原体。其中包括人类的非洲昏睡病（冈比亚锥虫和罗得西亚锥虫）和恰加斯病（克氏锥虫）以及牛的纳加纳病（刚果锥虫和布氏锥虫）。大量的临床和研究观察表明，这些寄生虫可能选择性地对还原氧代谢物超氧化物、过氧化氢和羟自由基的氧化应激敏感。这些后来的有毒物质可能是通过例如硝呋替莫和血卟啉等杀锥虫剂的单电子氧化还原循环产生的（Fairlamb，1982；Schirmer等人，1987）。 谷胱甘肽作为哺乳动物细胞中的主要硫醇，在多种细胞功能中发挥着不可或缺的作用，包括防止由谷胱甘肽过氧化物酶/谷胱甘肽进行的氧化应激还原酶酶对。尽管所有锥虫都表现出超氧化物歧化酶活性，但尚未发现铁依赖性过氧化物酶，因此表明谷胱甘肽依赖性过氧化物酶/还原酶对可能在寄生虫在宿主血流中的存活中发挥至关重要的作用。最近发现锥虫类含有很少的谷胱甘肽并且不含经典的谷胱甘肽还原酶，而是含有24元大环二硫键TV'。 A^-双谷胱甘肽亚精胺，考虑到微不足道
Revised Manuscript Received January 22, 1988 abstract: The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals> 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only thetwo arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GSt glutathione glycyl carboxylate, are absent. e trypanosomatid parasites are causative agents of a va-riety of serious diseases in both humansand domestic animals. These include African sleeping sickness (Trypanosoma gambiense and Trypanosoma rhodesiense) and Chagasdisease (Trypanosoma cruzi) in humans and nagana (Trypanosoma congolense and Trypanosoma brucei) in cattle. Numerous clinical andresearch observations indicatethat these parasites may be selectively susceptible to oxidant stress by the reduced oxygen metabolites superoxide, hydrogen peroxide, and hydroxyl radical. These later toxic species may be generated, for example, by one-electron redox cycling of such trypanocidal agents as nifurtimox and hematoporphyrin (Fairlamb, 1982; Schirmer et al., 1987).Glutathione, as the major thiol in mammalian cells, plays an integral role in a variety of cellular functions including the protection against oxidative stress as carried out by the glutathione peroxidase/glutathione reductase enzyme couple. Whereas all trypanosomatids show superoxide dismutase ac-tivity, no iron-dependent peroxidase has been discovered, thus suggesting that a glutathione-dependent peroxidase/reductase couple might play a crucial role in parasite survival in thehost blood stream. The recent discovery that trypanosomatids contain very little glutathione and no classical glutathione reductase but instead contain the 24-membered macrocyclic disulfide TV'. A^-bisiglutathionyljspermidine, given the trivial