Oza Sandra L, Tetaud Emmanuel, Ariyanayagam Mark R, Warnon Stephanie S, Fairlamb Alan H
School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
J Biol Chem. 2002 Sep 27;277(39):35853-61. doi: 10.1074/jbc.M204403200. Epub 2002 Jul 16.
Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.
动质体目原生动物与其他生物体的不同之处在于，它们能够将谷胱甘肽（L-γ-谷氨酰-半胱氨酰-甘氨酸）和亚精胺结合形成锥虫硫醇[N(1),N(8)-双(谷胱甘肽基)亚精胺]，这是一种参与抵御化学和氧化应激以及其他生物合成功能的代谢物。在fasiculata锥虫中，锥虫硫醇是由谷胱甘肽和亚精胺通过中间产物谷胱甘肽基亚精胺，分别在谷胱甘肽基亚精胺合成酶（GspS；EC ）和锥虫硫醇合成酶（TryS；EC ）催化的两个不同的ATP依赖性反应中合成的。在这里，我们从克氏锥虫中克隆了一个单拷贝基因（TcTryS），该基因编码的蛋白质与CfTryS的序列同一性为61%，但与CfGspS的序列同一性仅为31%。用TcTryS转化的酿酒酵母能够合成谷胱甘肽基亚精胺和锥虫硫醇，这表明该酶能够催化这两个生物合成步骤，这与CfTryS不同。当培养物中添加氨丙基尸胺时，酵母转化体中含有谷胱甘肽基氨丙基尸胺和同型锥虫硫醇[N(1),N(9)-双(谷胱甘肽基)氨丙基尸胺]，这些代谢物先前已在克氏锥虫中鉴定出，但在fasiculata锥虫中未发现。对从大肠杆菌中纯化的重组TcTryS的动力学研究表明，该酶对谷胱甘肽表现出高底物抑制（K(m)和K(i)分别为0.57和1.2 mM，k(cat)为3.4 s(-1)），但对亚精胺、氨丙基尸胺、谷胱甘肽基亚精胺和MgATP作为可变底物时遵循米氏动力学。重组酶具有较弱的酰胺酶活性，能够将锥虫硫醇、同型锥虫硫醇或谷胱甘肽基亚精胺水解为谷胱甘肽和相应的多胺。
