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Title: Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin

Abstract

The existence of natural peroxiredoxin-glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin module linked to glutaredoxin or thioredoxin modules. The recombinant fusion enzymes folded properly into non-covalently bound homodimers or homotetramers. Two of the three protein constructs exhibit peroxidase activity, a reaction where the two modules need to function together, but they also display enzymatic activities specific of each module. In addition, mass spectrometry analyses indicate that the Prx module can be both glutathiolated or overoxidized in vitro. This is discussed in the light of the Prx reactivity.

Authors:
 [1];  [2];  [3];  [2];  [4];  [2]
  1. UMR 1136 Interactions Arbres Microorganismes INRA UHP, IFR 110 GEEF, Faculte des Sciences, BP 239 54506 Vandoeuvre-les-Nancy Cedex (France). E-mail: nrouhier@scbiol.uhp-nancy.fr
  2. UMR 1136 Interactions Arbres Microorganismes INRA UHP, IFR 110 GEEF, Faculte des Sciences, BP 239 54506 Vandoeuvre-les-Nancy Cedex (France)
  3. Umea Plant Science Centre, Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology, SE-901 83 Umea (Sweden)
  4. Laboratoire de Resonance Magnetique Nucleaire, Institut de Biologie Structurale CEA-CNRS-UJF 'Jean-Pierre Ebel' 41 Avenue Jules Horowitz, 38027 Grenoble Cedex 1 (France)
Publication Date:
OSTI Identifier:
20798860
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 341; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2006.01.099; PII: S0006-291X(06)00192-6; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BACTERIA; BINDING ENERGY; HYBRIDIZATION; IN VITRO; MASS SPECTROSCOPY; PEROXIDASES; POPLARS; REACTIVITY

Citation Formats

Rouhier, Nicolas, Gama, Filipe, Wingsle, Gunnar, Gelhaye, Eric, Gans, Pierre, and Jacquot, Jean-Pierre. Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2006.01.099.
Rouhier, Nicolas, Gama, Filipe, Wingsle, Gunnar, Gelhaye, Eric, Gans, Pierre, & Jacquot, Jean-Pierre. Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin. United States. doi:10.1016/j.bbrc.2006.01.099.
Rouhier, Nicolas, Gama, Filipe, Wingsle, Gunnar, Gelhaye, Eric, Gans, Pierre, and Jacquot, Jean-Pierre. Fri . "Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin". United States. doi:10.1016/j.bbrc.2006.01.099.
@article{osti_20798860,
title = {Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin},
author = {Rouhier, Nicolas and Gama, Filipe and Wingsle, Gunnar and Gelhaye, Eric and Gans, Pierre and Jacquot, Jean-Pierre},
abstractNote = {The existence of natural peroxiredoxin-glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin module linked to glutaredoxin or thioredoxin modules. The recombinant fusion enzymes folded properly into non-covalently bound homodimers or homotetramers. Two of the three protein constructs exhibit peroxidase activity, a reaction where the two modules need to function together, but they also display enzymatic activities specific of each module. In addition, mass spectrometry analyses indicate that the Prx module can be both glutathiolated or overoxidized in vitro. This is discussed in the light of the Prx reactivity.},
doi = {10.1016/j.bbrc.2006.01.099},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 341,
place = {United States},
year = {Fri Mar 24 00:00:00 EST 2006},
month = {Fri Mar 24 00:00:00 EST 2006}
}
  • Yeast glutaredoxin 3 (Grx3) is a cytosolic protein that regulates the activity of the iron-responsive transcriptional activator Aft1. This member of the monothiol glutaredoxin family contains a thioredoxin-like domain and a glutaredoxin-like domain, which both possess a monothiol active site. The crystal structure of the thioredoxin-like domain has been determined at 1.5 {angstrom} resolution and represents the first published structure of this domain for the monothiol glutaredoxin family. The loop containing the signature motif WAxxC is partially disordered, indicating a greater degree of flexibility in this region compared with classical dithiol thioredoxins with a WCGPC active-site motif.
  • An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H{sub 2}O{sub 2}. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reducesmore » peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.« less
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  • Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold,more » in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.« less