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| Author(s): |
Bruno Brasil Horta
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2009-08-05 |
| Examining board members: |
Luis Eduardo Soares Netto;
Ohara Augusto;
Shaker Chuck Farah;
Fábio Cesar Gozzo;
Sandro Roberto Marana
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| Advisor: | Luis Eduardo Soares Netto |
| Abstract | |
The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of Citrus Variegated Chlorosis (CVC) that causes losses of about 100 millions dollars per year in Brazil. During infection, reactive oxygen species play a central role in plant pathogen defense. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including the peroxiredoxins alkyl hydroperoxide reductase subunit C (AhpC) and bacterioferritin comigratory protein (Bcp). Peroxiredoxins are peroxidases, which rely on an activated cysteine residue to catalyze the reduction of hydroperoxides. By proteome analysis, Smolka et al. (2003) identified the products of ahpc and bcp genes present in whole cell extract of X. fastidiosa. To characterize the function and structure of AhpC and Bcp protein, their genes were cloned in Escherichia coli and the corresponding proteins purified by nickel affinity chromatography. Recombinant proteins presented thiol-dependent peroxidase activity against hydrogen peroxide and organic hydroperoxides. AhpC and Bcp peroxidase activities are dependent on alkyl hydroperoxide reductase subunit F (AhpF), and on thioredoxin system, respectively. Paradoxically, AhpF flavoenzyme possesses hydrogen peroxide-forming oxidase activity. Contrary to classical assumptions, competitive kinetics employing horseradish peroxidase assays showed that the second-order rate constants of AhpC and Bcp reaction with hydrogen peroxide are in the order of 107 M-1.s-1, as fast as the activity of selenium-dependent glutathione peroxidases and catalases. Non-reducing SDS-PAGE and cysteine quantification using DTNB indicated different peroxidasic mechanisms: AhpC is a typical 2-Cys peroxiredoxin (with intermolecular disulfide bond formation), while Bcp is an atypical 2-Cys peroxiredoxin (with intramolecular disulfide bond formation). In contrast to the well-conserved AhpC cysteines responsible for the peroxidase activity (Cys-47 and Cys-165), only through site-specific mutagenesis and mass spectrometry we could identified the cysteine residues involved in the Bcp peroxidase activity (Cys-47 and Cys-83). Structural characterization by size exclusion chromatography and dynamic light scattering revealed that AhpC native protein forms stable and redox state independent decamers. The crystal structure of Bcp C47S, the first 2-Cys Prx with a 35-residue between the active cysteines ever characterized, shows that protein contains the common fold of peroxiredoxins and that active cysteines lies ~12.4 Å away one from the other. Based on circular dichroism, we presented data indicating that disulfide bond formation may require significant conformational changes, which probably is triggered by the peroxidatic cysteine oxidation to sulfenic acid. In conclusion, we elucidated the catalytic mechanisms and reduction systems of AhpC and Bcp proteins that may help to understand the pathogenicity mechanism of X. fastidiosa. These results can contribute to the development of plague control methods against X. fastidiosa. (AU) | |