A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi.
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.
Oza
Sandra L
SL
School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Tetaud
Emmanuel
E
Ariyanayagam
Mark R
MR
Warnon
Stephanie S
SS
Fairlamb
Alan H
AH
D000255:Adenosine Triphosphate / Q000378:metabolism; D019732:Amide Synthases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003421:Crithidia / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011073:Polyamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013095:Spermidine / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000201:enzymology*
D007477:Ions; D011073:Polyamines; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D004247:DNA; C044809:trypanothione; D017301:Ca(2+) Mg(2+)-ATPase; D019732:Amide Synthases; C081536:glutathionylspermidine synthetase; C064407:trypanothione synthetase; D005978:Glutathione; D013095:Spermidine