Leishmania major elongation factor 1B complex has trypanothione S-transferase and peroxidase activity.
In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified alpha, beta, and gamma subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg(-1)). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 mum for trypanothione and 7.4 mm for cumene hydroperoxide and kcat=25 s(-1). Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.
Vickers
Tim J
TJ
Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom.
Wyllie
Susan
S
Fairlamb
Alan H
AH
D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D018320:Leishmania major / Q000378:metabolism*; D008041:Linoleic Acids / Q000494:pharmacology; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D015262:Xenobiotics
D008041:Linoleic Acids; D008054:Lipid Peroxides; D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; D015262:Xenobiotics; C008285:linoleic acid hydroperoxide; D010544:Peroxidases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione