Location: Commodity Utilization ResearchTitle: Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria) Author
Submitted to: Advances in Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2012
Publication Date: 11/30/2012
Citation: Ullah, A.H.J., Sethumadhavan, K., Grimm, C., Shockey, J. 2012. Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from Lagenaria siceraria. Advances in Biological Chemistry. 2(4):403-410. Interpretive Summary: The biocatalyst, phosphatidic acid phosphohydrolase (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 (phosphatidic acid), the oleaginous plants won’t produce Triacylglyceride or TAG (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 CRIS is to engineer plants to produce industrial oil containing polyunsaturated fatty acids or PUFA so that the product could be used as drying oil. 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 from bottle gourd, which could hydrolyze the phosphate group from phosphatidic acid, a plant metabolite, and allows the oil synthesis to proceed. We also performed bioinformatics studies to find protein sequence similarities in other plants. This is the first reporting of this biocatalyst from an important vegetable that is known to produce oil in the mature seeds.
Technical Abstract: Phosphatidic acid phosphohydrolase (PAP), EC 188.8.131.52, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacyl glycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5 and 45ºC. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 nano Kat/mg of protein and 200 micro, respectively. For the synthetic substrate, p-nitrophenyl phosphate, p-NPP, the Vmax and Km were 33.0 nano Kat/mg of protein and 140 micro, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 1 to 10 mM. Two major protein bands at 42-KDa and 26-KDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of trypsinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolic enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.