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Biochim Biophys Acta Gen Subj. 2018 Mar;1862(3):775-789. doi: 10.1016/j.bbagen.2017.10.007. Epub 2017 Oct 12.

Disulfide bond formation protects Arabidopsis thaliana glutathione transferase tau 23 from oxidative damage.

Author information

1
VIB-VUB Center for Structural Biology, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
2
Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium; VIB Center for Medical Biotechnology, B-9000 Ghent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium; VIB Center for Plant Systems Biology, Technologiepark 927, B-9052 Ghent, Belgium.
3
Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium; VIB Center for Medical Biotechnology, B-9000 Ghent, Belgium.
4
Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium; VIB Center for Plant Systems Biology, Technologiepark 927, B-9052 Ghent, Belgium.
5
de Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium.
6
VIB-VUB Center for Structural Biology, B-1050 Brussels, Belgium; Brussels Center for Redox Biology, B-1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium. Electronic address: joris.messens@vib-vub.be.

Abstract

BACKGROUND:

Glutathione transferases play an important role as detoxifying enzymes. In A. thaliana, elevated levels of reactive oxygen species (ROS), provoked during biotic and abiotic stress, influence the activity of GSTU23. The aim of this study is to determine the impact of oxidative stress on the function and structure of GSTU23.

METHODS:

The impact of oxidation on the function of GSTU23 was studied using a glutathione transferase biochemical assay and mass spectrometry. With kinetics, circular dichroism and thermodynamics, we compared reduced with oxidized GSTU23. X-ray crystal structures of GSTU23 visualize the impact of oxidation on methionines and cysteines.

RESULTS:

In the presence of 100μM H2O2, oxidation of the methionine side-chain to a sulfoxide is the prominent post-translational modification, which can be reduced by C. diphtheriae MsrA and MsrB. However, increasing the level to 200μM H2O2 results in a reversible intramolecular disulfide between Cys65-Cys110, which is substrate for glutaredoxin. Under these oxidizing conditions, GSTU23 undergoes a structural change and forms a more favourable enzyme-substrate complex to overcome kcat decrease.

CONCLUSIONS AND SIGNIFICANCE:

At lower H2O2 levels (100μM), GSTU23 forms methionine sulfoxides. Specifically, oxidation of Met14, located near the catalytic Ser13, could interfere with both GSH binding and catalytic activation. At higher H2O2 levels (200μM), the Cys65-Cys110 disulfide bond protects other cysteines and also methionines from overoxidation. This study shows the impact of oxidative stress on GSTU23 regulated by methionine sulfoxide reductases and glutaredoxin, and the mechanisms involved in maintaining its catalytic functionality under oxidizing conditions.

KEYWORDS:

Disulfide bond; Glutathione transferase; Kinetics; Methionine sulfoxide; Thermodynamics; X-ray structure

PMID:
29031766
DOI:
10.1016/j.bbagen.2017.10.007
[Indexed for MEDLINE]

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