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Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

Iglesias-Baena I, Barranco-Medina S, Lázaro-Payo A, López-Jaramillo FJ, Sevilla F, Lázaro JJ - J. Exp. Bot. (2010)

Bottom Line: The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent.An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H).This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, E-18008 Granada, Spain.

ABSTRACT
The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

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Related in: MedlinePlus

Purification of recombinant AtSrx and confirmation of the Srx identity. (A) SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract; lane 2, after His-tag column; lane 3, pure protein after Mono Q chromatography. (B) Western blot with a specific antibody against Srx.
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fig2: Purification of recombinant AtSrx and confirmation of the Srx identity. (A) SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract; lane 2, after His-tag column; lane 3, pure protein after Mono Q chromatography. (B) Western blot with a specific antibody against Srx.

Mentions: A 375 pb cDNA was isolated from Arabidopsis thaliana RNA by RT-PCR and primers designed from the sequence of the putative AtSrx. The cDNA sequence encoded a 125 amino acids peptide with a molecular mass of 13 914 Da and a sequence-derived isoelectric point of 10.76, bearing a chloroplast transit peptide in the N-terminus and the signature sequence FG/SCHRY present in plant Srxs (Liu et al., 2006; Rey et al., 2007). A second PCR amplification with suitable primers led to the cDNA of the mature protein consisting of 103 amino acids with predicted molecular weight and isoelectric point of 11 546 and 9.86 Da, respectively. This cDNA was subcloned into pETM-11 expression vector and overexpressed in E. coli BL21 (DE3) to yield the recombinant N-terminal 6× His-tagged fusion mature Srx (AtSrx). The SDS-PAGE analysis showed that the His-tag approach led to a partially pure sample and additional Mono Q chromatography was needed to isolate the recombinant AtSrx (Fig. 2A). The Srx identity of the isolated recombinant protein was confirmed by Western blotting with antibodies against a Srx peptide and revealed a faint band at a molecular weight corresponding to a dimer of Srx (Fig. 2B).


Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

Iglesias-Baena I, Barranco-Medina S, Lázaro-Payo A, López-Jaramillo FJ, Sevilla F, Lázaro JJ - J. Exp. Bot. (2010)

Purification of recombinant AtSrx and confirmation of the Srx identity. (A) SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract; lane 2, after His-tag column; lane 3, pure protein after Mono Q chromatography. (B) Western blot with a specific antibody against Srx.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2837264&req=5

fig2: Purification of recombinant AtSrx and confirmation of the Srx identity. (A) SDS-PAGE and Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract; lane 2, after His-tag column; lane 3, pure protein after Mono Q chromatography. (B) Western blot with a specific antibody against Srx.
Mentions: A 375 pb cDNA was isolated from Arabidopsis thaliana RNA by RT-PCR and primers designed from the sequence of the putative AtSrx. The cDNA sequence encoded a 125 amino acids peptide with a molecular mass of 13 914 Da and a sequence-derived isoelectric point of 10.76, bearing a chloroplast transit peptide in the N-terminus and the signature sequence FG/SCHRY present in plant Srxs (Liu et al., 2006; Rey et al., 2007). A second PCR amplification with suitable primers led to the cDNA of the mature protein consisting of 103 amino acids with predicted molecular weight and isoelectric point of 11 546 and 9.86 Da, respectively. This cDNA was subcloned into pETM-11 expression vector and overexpressed in E. coli BL21 (DE3) to yield the recombinant N-terminal 6× His-tagged fusion mature Srx (AtSrx). The SDS-PAGE analysis showed that the His-tag approach led to a partially pure sample and additional Mono Q chromatography was needed to isolate the recombinant AtSrx (Fig. 2A). The Srx identity of the isolated recombinant protein was confirmed by Western blotting with antibodies against a Srx peptide and revealed a faint band at a molecular weight corresponding to a dimer of Srx (Fig. 2B).

Bottom Line: The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent.An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H).This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, E-18008 Granada, Spain.

ABSTRACT
The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

Show MeSH
Related in: MedlinePlus