Enzymatic properties of a recombinant phospholipid hydroperoxide glutathione peroxidase from Momordica charantia and its complementation function in yeast.
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 degrees 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.
Dong
Chun-Juan
CJ
Laboratory of Molecular Biology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China.
Yang
Xiao-Dong
XD
Liu
Jin-Yuan
JY
D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D005816:Genetic Complementation Test; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029777:Momordica charantia / Q000737:chemistry / Q000201:enzymology*; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D010940:Plant Proteins; D011994:Recombinant Proteins; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase