Cloning, expression and reconstitution of the trypanothione-dependent peroxidase system of Crithidia fasciculata

Emmanuel Tetaud, Alan H. Fairlamb (Lead / Corresponding author)

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    47 Citations (Scopus)

    Abstract

    As a consequence of aerobic metabolism, trypanosomatids are exposed to reactive oxygen intermediates such as superoxide, hydrogen peroxide and the hydroxyl radical. Metabolism of hydrogen peroxide in Crithidia fasciculata is accomplished by three distinct proteins, tryparedoxin, tryparedoxin peroxidase and trypanothione reductase, working in concert with the substrates NADPH and trypanothione. Here, we report the cloning and characterisation of the tryparedoxin (TryX) and tryparedoxin peroxidase (TryP) genes from C. fasciculata. Both genes are multicopy and organized in distinct tandem arrays in the genome. TryX encodes a 16 kDa protein, which belongs to the thioredoxin superfamily, sharing the WCPPC motif, whereas TryP encodes a 21 kDa protein belonging to a new class of peroxidases called 2-Cys peroxidoxins. Both TryX and TryP were expressed in Escherichia coli and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione and trypanothione reductase, similar to the native proteins. TryX is rapidly reduced by trypanothione, but weakly by glutathionylspermidine, glutathione or ovothiol A. TryP shows a broad substrate specificity and can reduced hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide with equal efficiency. Copyright (C) 1998 Elsevier Science B.V.

    Original languageEnglish
    Pages (from-to)111-123
    Number of pages13
    JournalMolecular and Biochemical Parasitology
    Volume96
    Issue number1-2
    DOIs
    Publication statusPublished - 30 Oct 1998

    Keywords

    • Drug design
    • Peroxidoxin
    • Thiol-specific antioxidant
    • Thioredoxin
    • Trypanothione peroxidase system

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