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Two Highly Homologous Methionine Sulfoxide Reductase A from Tomato (Solanum lycopersicum), Exhibit Distinct Catalytic Properties

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Abstract

E4, which is a fruit-ripening gene that is strongly induced by ethylene, has been reported to be a member of the methionine sulfoxide reductase A (MSRA) gene. In the present study, we determined for the first time the enzymatic activity and delineated the catalytic mechanism of the E4 protein via site-directed mutagenesis. The disulfide intermolecular cross-linking, kinetics parameter, thiol content titration analysis of wild-type and mutated E4 proteins revealed that the cysteine at position 37 (Cys-37) was the key catalytic residue, and Cys-194, but not Cys-180 served as the first recycling Cys in the thioredoxin (Trx)-dependent regeneration system. In addition, the SlMSRA2 protein, which was encoded by another MSRA gene, shared high similarity with the E4 protein and was truncated at the C-terminus. The wild-type and mutated SlMSRA2 enzymes had similar activities compared to the E4 protein using DTT as a reductant, but showed extremely low activities in the Trx-dependent reduction system. Our results indicated that E4 and SlMSRA2 proteins might exhibit distinct catalytic mechanisms.

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Abbreviations

Dabsyl:

4-(Dimethylamino)azobenzene-4′-sulfonyl chloride

Dabsyl-Met:

Dabsyl-Cl labelled methionine

Dabsyl-MetSO:

Dabsyl-Cl labelled methionine sulfoxide

DTNB:

5,5′-Dithiobis-2-nitrobenzoic acid

DTT:

Dithithreitol

HPLC:

High performance liquid chromatography

MetSO:

Methionine sulfoxide

MSR:

Methionine sulfoxide reductase

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

Trx:

Thioredoxin

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Acknowledgments

This work was partially supported by a grant from Institute of Biosciences and Biotechnology at Kangwon National University. We thank Prof. Pascal Rey and Dr. Lionel Tarrago for technical assistance.

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Authors and Affiliations

  1. Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon-do, 200-701, South Korea

    Changbo Dai, Woong Han & Myeong-Hyeon Wang

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  1. Changbo Dai
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  2. Woong Han
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  3. Myeong-Hyeon Wang
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Corresponding author

Correspondence to Myeong-Hyeon Wang.

Additional information

Changbo Dai and Woong Han have contributed equally to this work.

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Dai, C., Han, W. & Wang, MH. Two Highly Homologous Methionine Sulfoxide Reductase A from Tomato (Solanum lycopersicum), Exhibit Distinct Catalytic Properties. Protein J 31, 285–292 (2012). https://doi.org/10.1007/s10930-012-9403-z

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