Identification of an Mg2+-Independent Soluble Phosphatidate Phosphatase in Cottonseed (Gossypium hirsutum L.)

Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP 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. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg2+-independent soluble PAP activity was identified from the 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 pH 5 and 55℃, respectively. Under optimized assay conditions, the Vmax and Km values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/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 did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.

Measuring phosphatidic acid phosphohydrolase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods

Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthophosphate. In eukaryotes, the PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG). Existing methods for measuring PAP activity rely on the use of radioactive PtdOH. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a coproduct of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 ηkat per mg of protein. Thus, it is now possible to perform PAP activity using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.

A Single Mutation in the Hepta-Peptide Active Site of <i>Aspergillus niger</i>PhyA Phytase Leads to Myriad Biochemical Changes

The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.

Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from <i>Lagenaria siceraria</i>

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.

Elimination of a disulfide bridge in <i>Aspergillus niger</i>NRRL 3135 Phytase (PhyA) enhances heat tolerance and optimizes its temperature versus activity profile

In this study, the optimum temperature was lowered while the residual phytase activity after heating to 70℃ was raised in a widely utilized phytase, Aspergillus niger NRRL 3135 PhyA. This was accomplished by site-directed mutagenesis of the cysteines that are involved in the formation of a single disulfide bridge (DB). When compared to wild type (WT), three of the four mutant phytases displayed a lower optimum temperature, 42℃, and up to a four-fold increase in activity after heating. These findings have a potentially broad application to be incorporated along with other desirable features to engineer a phytase with superior physical and chem-ical attributes for animal feed applications.

Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of <i>Momordica</i><i>charantia</i>

Phosphatidate phosphatase (EC 3.1.3.4), PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol. A Mg2+ independent PAP activity was identified in the soluble extract of Momordica charantia cotyledons undergoing maturation. While the microsomal fraction of the extract gave only 10% of the PAP activity, the remaining 90% of the activity was associated with the soluble fraction. At pH 3.0, the soluble PAP was bound to S column and eluted with glycine-HCl buffer containing high salt. The pH and temperature optima of the PAP activity were 6.0 and 53℃, respectively. Under optimum assay condition, the Vmax and Km for dioleoyl phosphatidic acid were 1.89 ηkat/mg of protein and 142 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ- NPP, the Vmax and Km were 10.4 ηkat/mg of protein and 107 μM, respectively. The inclusion of Mg2+ and β-mercaptoethanol into the reaction mix did not change the enzyme activity nor did the addition of N-ethylmaleimide and phenylglyoxal, which indicates that cysteine and arginine are not involved in catalysis of PtdOH. The addition of Mg2+ up to 10 mM also did not change the level of PAP activity. Triton X-100, however, inhibited the activity. This is the first documented case of an in vitro PAP activity in the developing cotyledons of Momordica charantia. The PAP described here could serve as a model for lipin-1 or lipin-2 in humans. Mutations in these genes lead to acute myoglobinuria in human infants.

Ethanol Tolerance in <i>Aspergillus niger</i>and <i>Escherichia coli</i>Phytase

Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.