Submitted to: Advances in Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 26, 2012
Publication Date: February 17, 2013
Citation: Ullah, A.H.J., Sethumadhavan, K. 2013. Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of Morordica charantia. Advances in Biological Chemistry. (3):11-17. Interpretive Summary: The biocatalyst, phosphatidic acid phosphatase (PAP), performs a committed step in the production of oil in the seed of oil-producing crops such as soybean, cottonseed, peanut, etc. If the biocatalyst (enzyme) functions sub-optimally because of poor recognition of the substrate or fatty acids, the oleaginous plants won’t produce oil efficiently. The enzyme works at the penultimate step of the Kennedy Pathway, which is responsible for oil synthesis in plants seeds. One of the goals of our Project is to engineer plants to produce oil containing polyunsaturated fatty acids or PUFA. To achieve this goal, we have to make sure that PAP could recognize and accept PUFA as a substrate for PAP. Therefore, we need to study this biocatalyst while determining the enzymatic properties including the range of substrates this enzyme could accommodate into the catalytic site or the engine of the enzyme. We report here a functional PAP enzyme, which could hydrolyze the phosphate group from phosphatidic acid, a plant metabolite, and allows the oil synthesis to proceed. This is the first reporting of this biocatalyst from an important vegetable that is known to produce heavy oil in the mature seeds.
Technical Abstract: Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 22.214.171.124), which is also known as PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol (DAG) and inorganic phosphate. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG), which is a storage lipid. The DAG is also a precursor to phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) that are constituents of membrane lipids. A Mg++ independent PAP activity was identified and characterized from the soluble extract made from maturing cotyledons of bitter gourd, Momordica charantia. The pH and temperature optima of the PAP activity were 6.0 and 53 ºC, respectively. Under optimum assay condition, for the substrate dioleoyl phosphatidic acid (DPA), the Vmax and Km were 1.89 ' kat/mg of protein and 142 µM, respectively. For the synthetic substrate, '-nitrophenylphosphate, '-NPP, the Vmax and Km were 10.4 'kat/mg of protein and 107 µM, respectively. The inclusion of Mg++ and ß-mercaptoethanol into the reaction mix did not change the enzyme activity, nor did the addition of N-ethylmaleimide and phenylglyoxal indicating that cysteine and arginine are not involved in catalysis of PtdOH. The addition of Mg++ up to 10 mM also did not change the level of PAP activity. The addition of incremental increase of Triton X-100 to the reaction mix inhibited the activity. This is the first documented case of an in vitro PAP activity in the developing cotyledons of Momordica charantia, which produces heavy oil in the seed that contains a-eleostearic acid (9,11,13-octadecatrienoic acid) as acyl chain.