The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.
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Authors
Trujillo, MadiaMauri, PierLuigi
Benazzi, Louise
Comini, Marcelo
De Palma, Antonella
Flohé, Leopold
Radi, Rafael
Stehr, Matthias
Singh, Mahavir
Ursini, Fulvio
Jaeger, Timo
Issue Date
2006-07-21
Metadata
Show full item recordAbstract
Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.Citation
The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. 2006, 281 (29):20555-66 J. Biol. Chem.Affiliation
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, UY-11800 Montevideo, Uruguay.PubMed ID
16682410Type
ArticleLanguage
enISSN
0021-9258ae974a485f413a2113503eed53cd6c53
10.1074/jbc.M601008200
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