Tryparedoxin Peroxidase of Leishmania donovani: Molecular Cloning, Heterologous Expression, Specificity, and Catalytic Mechanism |
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Authors: | Leopold Flohé Heike BuddeKarsten Bruns Helena CastroJoachim Clos Birgit HofmannSonia Kansal-Kalavar Dirk KrummeUlrich Menge Karin Plank-SchumacherHelena Sztajer Joseph WissingClaudia Wylegalla Hans-Jürgen Hecht |
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Institution: | a Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, D-38124, Braunschweig, Germanyb Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124, Braunschweig, Germanyc University of Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugald Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20539, Hamburg, Germany |
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Abstract: | Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H2O2, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a kcat of 11.2 s−1 and limiting Km values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 μM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173′ that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis. |
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Keywords: | tryparedoxin peroxidase Leishmania donovani peroxiredoxin site-directed mutagenesis kinetics catalytic mechanism molecular models |
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