title: Tryparedoxin peroxidase of Leishmania donovani: molecular cloning, heterologous expression, specificity, and catalytic mechanism.
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 k(cat) of 11.2 s(-1) and limiting K(m) values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 microM, 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.
authors.affiliation: Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, Braunschweig, D-38124, Germany. lfl@GBF.de
authors.forename: Leopold
authors.initials: L
authors.lastname: Flohé
authors.forename: Heike
authors.initials: H
authors.lastname: Budde
authors.forename: Karsten
authors.initials: K
authors.lastname: Bruns
authors.forename: Helena
authors.initials: H
authors.lastname: Castro
authors.forename: Joachim
authors.initials: J
authors.lastname: Clos
authors.forename: Birgit
authors.initials: B
authors.lastname: Hofmann
authors.forename: Sonia
authors.initials: S
authors.lastname: Kansal-Kalavar
authors.forename: Dirk
authors.initials: D
authors.lastname: Krumme
authors.forename: Ulrich
authors.initials: U
authors.lastname: Menge
authors.forename: Karin
authors.initials: K
authors.lastname: Plank-Schumacher
authors.forename: Helena
authors.initials: H
authors.lastname: Sztajer
authors.forename: Joseph
authors.initials: J
authors.lastname: Wissing
authors.forename: Claudia
authors.initials: C
authors.lastname: Wylegalla
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003001:Cloning, Molecular; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D010545:Peroxides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; C110959:tryparedoxin peroxidase