Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia)

Authors: Cao, Heping; Sethumadhavan, Kandan; Grimm, Casey; Ullah, Abul

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/2014
Publication Date: 9/9/2014
Citation: Cao, H., Sethumadhavan, K., Grimm, C.C., Ullah, H.J. 2014. Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia). PLoS One. 9(9):e106403.
DOI: https://doi.org/10.1371/journal.pone.0106403

Interpretive Summary: Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. It is a tropical and subtropical vine of the Cucurbitaceae family and widely grown in Asia, Africa and the Caribbean. Bitter melon seeds are rich in fatty acids and minerals including iron, beta carotene, calcium, potassium and many vitamins. Bitter melon is often used in Chinese cooking for its bitter flavor, typically in stir-fries, soups and herbal teas. It has also been used as the bitter ingredient in some Chinese and Okinawan beers. Bitter melon has also been used as herbal medicine in Asian and African for a long time. Recent studies have demonstrated the potential uses of bitter melon oil with a wide range of nutritional and medicinal applications. Alpha-eleostearic acid (a-ESA) counts for over half of the total fatty acids in bitter melon seeds, but little is known about the enzymatic mechanism for the biosynthesis of a-ESA. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. In eukaryotes, PAP-driven reaction is a committed step in the synthesis of triacylglycerol. PAPs are widely present in plants, animals, microbes and human. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the purification and characterization of PAP from developing cotyledons of bitter melon. Our results suggest that PAP protein is associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants.

Technical Abstract: Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg2+-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 50-65 °C and unaffected by up to 0.3 mM MgCl2. The Km and Vmax values for DPA were 595.4 µM and 104.9 'kat/mg of protein, respectively. PAP activity was inhibited by NaF, Na3VO4, Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants.