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A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies

Authors

  • X.-W. Chi,

    1.  Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
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    • These authors contributed equally to the paper.

  • C.-T. Lin,

    1.  Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan
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    • These authors contributed equally to the paper.

  • Y.-C. Jiang,

    1.  Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
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    • These authors contributed equally to the paper.

  • L. Wen,

    1.  Department of Chemistry, Western Illinois University, Macomb, IL, USA
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    • These authors contributed equally to the paper.

  • C.-T. Lin

    1.  Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
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  • Editor
    E. Flemetakis

C.-T. Lin, Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung 202, Taiwan.
E-mail: B0220@mail.ntou.edu.tw

Abstract

Glutaredoxins (Grx) play an important role in reduction of protein glutathione mixed disulphides. An IbGrx cDNA (561 bp, EF362614) encoding a putative dithiol Grx was cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grx, having a CGYC dithiol motif at the active site. A 3-D structural model was created based on the known crystal structure of a poplar Grx (GrxC1). To characterise the IbGrx protein, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His6-tagged IbGrx was expressed and purified by metal affinity chromatography. The purified enzyme showed a monomeric band, as demonstrated with 15% SDS-PAGE. The Michaelis constant (KM) for ß-hydroxyethyl disulphide (HED) was 0.50 ± 0.08 Mm. The enzyme retained 60% activity at 80 °C for 16 min. The enzyme was active over a broad pH range from 6.0 to 11.0, and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to protease.

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