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PMID:20335172

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Citation

Horta, BB, de Oliveira, MA, Discola, KF, Cussiol, JR and Netto, LE (2010) Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity. J. Biol. Chem. 285:16051-65

Abstract

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Links

PubMed PMC2871474 Online version:10.1074/jbc.M109.094839

Keywords

Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Catalysis; Crystallography, X-Ray; Disulfides/chemistry; Dithiothreitol/chemistry; Hydrogen Peroxide/chemistry; Kinetics; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Peroxiredoxins/chemistry; Peroxiredoxins/genetics; Peroxynitrous Acid/chemistry; Protein Structure, Secondary; Structure-Activity Relationship; Xylella/enzymology; Xylella/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

Notes

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References

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