Structural and Biochemical Characterization of Peroxiredoxin Qβ from Xylella fastidiosa

• Published in: The Journal of biological chemistry 2010-05, Vol.285 (21), p.16051-16065
• Main Authors: Horta, Bruno Brasil, de Oliveira, Marcos Antonio, Discola, Karen Fulan, Cussiol, José Renato Rosa, Netto, Luis Eduardo Soares
• Format: Article
• Language: English
• Subjects: Enzyme Kinetics; Enzyme Mechanisms; Peroxidase; Peroxiredoxin; Protein Structure; Thiol; Thioredoxin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.094839

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 107 and 106m−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 ∼12.3 Å 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 α-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.