Tetaud E, Manai F, Barrett M P, Nadeau K, Walsh C T, Fairlamb A H
Department of Biochemistry, Wellcome Trust Building, University of Dundee Dundee DD1 4HN, Scotland, United Kingdom.
J Biol Chem. 1998 Jul 31;273(31):19383-90. doi: 10.1074/jbc.273.31.19383.
Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (gamma-Glu-Cys-Gly) and spermidine to form trypanothione (N1,N8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.
动质体目原生动物与其他生物的不同之处在于，它们能够将谷胱甘肽（γ-谷氨酰-半胱氨酰-甘氨酸）和亚精胺结合形成锥虫硫醇（N1,N8-双（谷胱甘肽基）亚精胺），这参与维持细胞内硫醇氧化还原状态以及抵御氧化剂。在本研究中，来自 fasciculata 克氏锥虫的 Cf-GSS 和 Cf-TRS 基因已被克隆并表达，这两个基因分别编码谷胱甘肽基亚精胺合成酶（EC 6.3.1.8）和锥虫硫醇合成酶（EC 6.3.1.9）。Cf-GSS 和 Cf-TRS 的推导氨基酸序列与大肠杆菌谷胱甘肽基亚精胺合成酶/酰胺酶具有 50%的序列相似性。这两个基因在 fasciculata 克氏锥虫基因组中均以单拷贝形式存在。当在大肠杆菌和酿酒酵母中表达时，两种蛋白质均未以活性可溶性形式存在。然而，对酿酒酵母的硫醇分析表明，用 Cf-GSS 基因转化的细胞含有大量谷胱甘肽基亚精胺，而同时表达 Cf-GSS 和 Cf-TRS 基因的细胞含有谷胱甘肽基亚精胺和锥虫硫醇，这证实这些基因编码来自 fasciculata 克氏锥虫的功能性谷胱甘肽基亚精胺和锥虫硫醇合成酶。Cf-GSS 和 Cf-TRS 的翻译产物与大肠杆菌谷胱甘肽基亚精胺合成酶中存在的酰胺酶结构域具有显著同源性，该结构域可催化谷胱甘肽基亚精胺的合成与降解。从 fasciculata 克氏锥虫中分离出的谷胱甘肽基亚精胺合成酶被发现具有类似的酰胺酶活性。
