Identification of an Mg2+-independent soluble phosphatidate phosphatase in cottonseed (Gossypium hirsutum L.)

Authors: Cao, Heping; Sethumadhavan, Kandan; Rajasekaran, Kanniah - Rajah

Submitted to: Advances in Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2016
Publication Date: 11/21/2016
Citation: Cao, H., Sethumadhavan, K., Rajasekaran, K. 2016. Identification of an Mg2+-independent soluble phosphatidate phosphatase in cottonseed (Gossypium hirsutum L.). Advances in Biological Chemistry. 6(6):169-179.
DOI: https://doi.org/10.4236/abc.2016.66015

Interpretive Summary: Cotton plant provides two economically important products, fiber as the major product and cottonseed as the minor product. However, cotton plant produces more seed in terms of quantity than fiber with a seed/fiber ratio of 1.5 - 1.7. Cottonseed typically accounts for 15 - 25% of the crop value. Oil is the most valuable product of the cottonseed and used primarily as food or food ingredient. Cottonseed oil is rich in linoleic acid, a polyunsaturated fatty acid (~54%) and relatively low in oleic acid, a monosaturated fatty acid (~16%). This composition of fatty acids allows the oil to be useful for some particular applications, such as for salad dressings and cooking oil. However, the high ratio of polyunsaturates to monounsaturates results in reduced oxidative stability compared with other cooking oils. Currently, much needs to be learned about the enzymatic mechanism of the oil biosynthesis in cottonseed. Towards understanding the biochemical pathway of oil accumulation in cottonseed, we initially focused our studies on PAP (3-sn-phosphatidate phosphohydrolase) that dephosphorylates phosphatidic acid to generate diacylglycerol and inorganic phosphate. PAP family enzymes are currently classified as either soluble PAP or membrane-bound PAP. Based on the requirement of Mg2+ for activity, PAP could also be divided into 2 classes: Mg2+-dependent and Mg2+-independent PAP. Typically, soluble PAP is Mg2+-dependent; whereas membrane-bound PAP is Mg2+-independent. We recently identified and characterized a soluble PAP in bitter melon cotyledons. We report here the identification and partial characterization of PAP from developing cottonseed as a soluble and Mg2+-independent enzyme.

Technical Abstract: Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the oil biosynthesis pathway in cottonseed. Towards understanding the biochemical pathway of oil accumulation in cottonseed, this study focused on 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 generally categorized into Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the identification of an Mg2+-independent soluble PAP activity from developing cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were 5 and 55°C, respectively. Under optimum assay conditions, the Vmax and Km for dioleoyl phosphatidic acid as the substrate were 2.8 'kat/mg of protein and 539 µM, respectively. Inclusion of the detergent Triton X-100 (0 - 0.5%) or magnesium chloride (1 mM) in the reaction mix didn't alter activity to a significant degree. This is the first report of a PAP activity in the developing seeds of Gossipium hirsutum.