title: A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata.
abstract: Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases. The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione. Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert. One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides. By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated. Cf21 proved to be a moderately efficient peroxidase with broad specificity. The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively. The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1. This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.
authors.affiliation: GBF-Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Nogoceke
authors.forename: D U
authors.initials: DU
authors.lastname: Gommel
authors.forename: M
authors.initials: M
authors.lastname: Kiess
authors.forename: H M
authors.initials: HM
authors.lastname: Kalisz
authors.forename: L
authors.initials: L
authors.lastname: Flohé
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016831:Crithidia fasciculata / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D013879:Thioredoxins; C044809:trypanothione; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005978:Glutathione; D013095:Spermidine