Pre-steady state kinetic characterization of human peroxiredoxin 5: Taking advantage of Trp84 fluorescence increase upon oxidation

Trujillo, Madia; Clippe, André; Manta, Bruno; Ferrer-Sueta, Gerardo; Smeets, Aude; Declercq, Jean‐Paul; Knoops, Bernard; Radí, Rafael

doi:10.1016/j.abb.2007.08.008

Cited by 159 publications

(194 citation statements)

References 58 publications

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“…Again, this finding is consistent with the proposal that the recycling step of Prx reduction limits the catalytic cycle (21)(22)(23)51). This result can explain why DTTdependent peroxidase activity of XfPrxQ C83S was higher than wild-type XfPrxQ activity (Fig.…”

Section: Discussionsupporting

confidence: 81%

“…It is important to mention that cleavage of the His tag from recombinant fusion proteins did not change considerably their activities (data not shown). New kinetic approaches have revealed that class 3 Prx and class 4 Prx enzymes present high reactivity toward hydroperoxides (21)(22)(23)51), changing the view that Prx enzymes are only moderately reactive. Because this kind of analysis had not yet been performed for class 1 Prx enzymes, the second-order rate constant of the reaction between reduced XfPrxQ and hydrogen peroxide was measured by competitive assay (22).…”

Section: Resultsmentioning

confidence: 99%

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Structural and Biochemical Characterization of Peroxiredoxin Qβ from Xylella fastidiosa

Horta

Oliveira

Discola

et al. 2010

Journal of Biological Chemistry

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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 ϳ12.3 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 ␣-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.

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Section: Discussionsupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Structural and Biochemical Characterization of Peroxiredoxin Qβ from Xylella fastidiosa

Horta

Oliveira

Discola

et al. 2010

Journal of Biological Chemistry

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“…In fact, the rate constant of peroxynitrite reduction by Ohr is comparable to the rate constants reported for Prxs and thiolor selenol-dependent Gpxs (10,12,13,(53)(54)(55). Therefore, it is feasible that Ohr plays a relevant role in vivo in the reduction of peroxynitrite.…”

Section: Discussionsupporting

confidence: 52%

“…According to the Brønsted relationship, the pK a of the leaving group is a factor that is involved in the reactivity of thiolates with hydroperoxides (13). The pK a values for aliphatic alkoxyl groups, which are the leaving groups corresponding to t-BHP and fatty acid hydroperoxides, are expected to have similar values (13).…”

Section: Discussionmentioning

confidence: 99%

“…The pK a values for aliphatic alkoxyl groups, which are the leaving groups corresponding to t-BHP and fatty acid hydroperoxides, are expected to have similar values (13). Therefore, it is reasonable to think that the reactions of t-BHP and fatty acid hydroperoxides with ordinary thiols (e.g., the thiol of BSA) are within the same range and that the catalytic power of Ohr proteins for fatty acid hydroperoxides is therefore extraordinary (at least 10 6 -fold increase).…”

Section: Discussionmentioning

confidence: 99%

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Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Alegria

Meireles

Cussiol

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Organic hydroperoxide resistance (Ohr) enzymes are unique Cysbased, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 10 7 -10 8 M −1 ·s −1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 10 7 M −1 ·s −1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a Δohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the Δohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the Δohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H 2 O 2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wildtype strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.hydroperoxides | thiols | Cys-based peroxidase | pathogenic bacteria | Pseudomonas aeruginosa

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Overview of Peroxiredoxins in Oxidant Defense and Redox Regulation

2011

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Peroxiredoxins are important hydroperoxide detoxification enzymes, yet have only come to the fore in recent years relative to the other major players in peroxide detoxification, heme-containing catalases and peroxidases, and glutathione peroxidases. These cysteine-dependent peroxidases exhibit high reactivity with hydrogen peroxide, organic hydroperoxides and peroxynitrite and play major roles not only in peroxide defense, but also in regulating peroxide-mediated cell signaling. This overview focuses on important peroxiredoxin features that have emerged over the past several decades with an emphasis on catalytic mechanism, regulation and biological function.

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Pre-steady state kinetic characterization of human peroxiredoxin 5: Taking advantage of Trp84 fluorescence increase upon oxidation

Cited by 159 publications

References 58 publications

Structural and Biochemical Characterization of Peroxiredoxin Qβ from Xylella fastidiosa

Structural and Biochemical Characterization of Peroxiredoxin Qβ from Xylella fastidiosa

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Overview of Peroxiredoxins in Oxidant Defense and Redox Regulation

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