title: Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.
abstract: Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Vickers
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D016831:Crithidia fasciculata / Q000201:enzymology; D003432:Cross-Linking Reagents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits; D012269:Ribosomal Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014227:Triazines / Q000494:pharmacology; D014351:Trypanosomatina / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D020648:Peptide Elongation Factor 1; D021122:Protein Subunits; D012269:Ribosomal Proteins; D014227:Triazines; C036598:procion blue MX-R; C012842:Cibacron Blue F 3GA; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005982:Glutathione Transferase; D019704:Protein Disulfide-Isomerases