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Enzymatic properties of a recombinant phospholipid hydroperoxide glutathione peroxidase from Momordica charantia and its complementation function in yeast

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Abstract

The entire encoding region for Momordica charantia phospholipid hydroperoxide glutathione peroxidase (McPHGPx) was cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The purified recombinant McPHGPx displayed GSH-dependent peroxidase activity towards phospholipid hydroperoxide, H2O2, and tert-butyl hydroperoxide and had the highest affinity with and catalytic efficiency for phospholipid hydroperoxide. The optimum temperature of the enzyme activity ranged from 40 to 50°C, thus it is a thermostable enzyme compared to other PHGPx enzymes. Furthermore, McPHGPx expression in Saccharomyces cerevisiae PHGPx-deletion mutant rescued the susceptibilities to the oxidation-sensitive polyunsaturated fatty acid (linolenic acid), indicating its PHGPx complementation function in yeast. These results have well documented that McPHGPx functions as a PHGPx in vitro and in vivo and will be beneficial for further functional studies on plant PHGPx enzymes.

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Abbreviations

BHT:

butylated hydroxytoluene

GPx:

glutathione peroxidase

IPTG:

isopropyl β-D-thiogalactopyranoside

LA:

linolenic acid

(Mc)PHGPx:

(Momordica charantia) phospholipid hydroperoxide glutathione peroxidase

MDA:

malondialdehyde

PCOOH:

phosphatidylcholine hydroperoxide

ROS:

reactive oxygen species

SD/Trp− :

synthetic complete medium without tryptophan

TBA:

thiobarbituric acid

t-BHP:

tert-butyl hydroperoxide

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Authors and Affiliations

  1. Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China

    Chun-Juan Dong, Xiao-Dong Yang & Jin-Yuan Liu

Authors
  1. Chun-Juan Dong
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  2. Xiao-Dong Yang
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  3. Jin-Yuan Liu
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Correspondence to Jin-Yuan Liu.

Additional information

Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 620–628.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM08-310, February 15, 2009.

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Dong, CJ., Yang, XD. & Liu, JY. Enzymatic properties of a recombinant phospholipid hydroperoxide glutathione peroxidase from Momordica charantia and its complementation function in yeast. Biochemistry Moscow 74, 502–508 (2009). https://doi.org/10.1134/S0006297909050046

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  • DOI: https://doi.org/10.1134/S0006297909050046

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