Abstract
Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (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?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing 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.
Highlights
Phosphatidic acid phosphohydrolase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) hydrolyzes the phosphomonoester bond present in phosphatidate (PtdOH) yielding diacylglycerol (DAG) and Pi
The reaction volume was fixed at 1.0 mL to which was added 50 mM sodium acetate, pH 5.5, 25 to 50 L of samples containing L. siceraria PAP enzyme followed by 50 L of 10 mM dioleoyl phosphatidic acid to start the enzymatic reaction
It is worth noting here that the flow through of Q column had turbidity and very little PAP activity associated with it. This indicates that microsomal fraction of the preparation had a negligible PAP activity. This bolsters our notion that we have identified a soluble PAP activity in the cotyledon extract of Lagenaria siceraria
Summary
Phosphatidic acid phosphohydrolase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) hydrolyzes the phosphomonoester bond present in phosphatidate (PtdOH) yielding diacylglycerol (DAG) and Pi. The kinetic and other physico-chemical data of this key biocatalyst from oleaginous plants is deemed useful for further study of plant fatty acid synthesis systems and for the development of novel and alternative oil sources. Such studies may provide insights into processes for further modification, enhancement, and control the total fatty acyl composition of TAG in oil-producing plants. We characterized L. siceraria PAP as detected in the crude supernatant derived from developing cotyledons by measuring activity, conducting kinetics experiments, and determining pH optima, temperature optima, and effects of minerals and other reagents of this key biocatalyst. This is the first documented reporting of PAP activity in L. siceraria
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