2.50
Hdl Handle:
http://hdl.handle.net/10033/15372
Title:
The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.
Authors:
Trujillo, Madia; Mauri, PierLuigi; Benazzi, Louise; Comini, Marcelo; De Palma, Antonella; Flohé, Leopold; Radi, Rafael; Stehr, Matthias; Singh, Mahavir; Ursini, Fulvio; Jaeger, Timo
Abstract:
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.
Affiliation:
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, UY-11800 Montevideo, Uruguay.
Citation:
The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. 2006, 281 (29):20555-66 J. Biol. Chem.
Journal:
The Journal of biological chemistry
Issue Date:
21-Jul-2006
URI:
http://hdl.handle.net/10033/15372
DOI:
10.1074/jbc.M601008200
PubMed ID:
16682410
Type:
Article
Language:
en
ISSN:
0021-9258
Appears in Collections:
publications of the research group genomeanalytics (GMAK)

Full metadata record

DC FieldValue Language
dc.contributor.authorTrujillo, Madia-
dc.contributor.authorMauri, PierLuigi-
dc.contributor.authorBenazzi, Louise-
dc.contributor.authorComini, Marcelo-
dc.contributor.authorDe Palma, Antonella-
dc.contributor.authorFlohé, Leopold-
dc.contributor.authorRadi, Rafael-
dc.contributor.authorStehr, Matthias-
dc.contributor.authorSingh, Mahavir-
dc.contributor.authorUrsini, Fulvio-
dc.contributor.authorJaeger, Timo-
dc.date.accessioned2007-12-18T09:21:20Z-
dc.date.available2007-12-18T09:21:20Z-
dc.date.issued2006-07-21-
dc.identifier.citationThe mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin. 2006, 281 (29):20555-66 J. Biol. Chem.en
dc.identifier.issn0021-9258-
dc.identifier.pmid16682410-
dc.identifier.doi10.1074/jbc.M601008200-
dc.identifier.urihttp://hdl.handle.net/10033/15372-
dc.description.abstractThioredoxin 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.en
dc.language.isoenen
dc.subject.meshBacterial Proteinsen
dc.subject.meshCatalytic Domainen
dc.subject.meshCysteineen
dc.subject.meshDNA Primersen
dc.subject.meshKineticsen
dc.subject.meshMutagenesis, Site-Directeden
dc.subject.meshMycobacterium tuberculosisen
dc.subject.meshOxidation-Reductionen
dc.subject.meshPeroxidasesen
dc.subject.meshPeroxiredoxinsen
dc.subject.meshProtein Conformationen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshX-Ray Diffractionen
dc.titleThe mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.en
dc.typeArticleen
dc.contributor.departmentDepartamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, UY-11800 Montevideo, Uruguay.en
dc.identifier.journalThe Journal of biological chemistryen

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