Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

Molecular properties of the class III subfamily of acyl-coenyzme A binding proteins from tung tree (Vernicia fordii).

Acyl-CoA binding proteins (ACBPs) have been identified in most branches of life, and play various roles in lipid metabolism, among other functions. Plants contain multiple classes of ACBP genes. The most diverse group is the class III proteins. Tung tree (Vernicia fordii) contains two such genes, designated VfACBP3A and VfACBP3B. The two proteins are significantly different in length and sequence. Analysis of tung ACBP3 genes revealed significant evolution, suggesting relatively ancient divergence of the two genes from a common ancestor. Phylogenetic comparisons of multiple plant class III proteins suggest that this group is the most evolutionarily dynamic class of ACBP. Both tung ACBP3 genes are expressed at similar levels in most tissues tested, but ACBP3A is stronger in leaves. Three-dimensional modeling predictions confirmed the presence of the conserved four α-helix bundle acyl-CoA binding (ACB); however, other regions of these proteins likely fold much differently. Acyl-CoA binding assays revealed different affinities for different acyl-CoAs, possibly contradicting the redundancy of function suggested by the gene expression studies. Subcellular targeting of transiently-expressed plant ACBP3 proteins contradicted earlier studies, and suggested that at least some class III ACBPs may be predominantly targeted to endoplasmic reticulum membranes, with little or no targeting to the apoplast.

Phytase, a new life for an "old" enzyme.

Phytases are phosphohydrolytic enzymes that initiate stepwise removal of phosphate from phytate. Simple-stomached species such as swine, poultry, and fish require extrinsic phytase to digest phytate, the major form of phosphorus in plant-based feeds. Consequently, this enzyme is supplemented in these species' diets to decrease their phosphorus excretion, and it has emerged as one of the most effective and lucrative feed additives. This chapter provides a comprehensive review of the evolving course of phytase science and technology. It gives realistic estimates of the versatile roles of phytase in animal feeding, environmental protection, rock phosphorus preservation, human nutrition and health, and industrial applications. It elaborates on new biotechnology and existing issues related to developing novel microbial phytases as well as phytase-transgenic plants and animals. And it targets critical and integrated analyses on the global impact, novel application, and future demand of phytase in promoting animal agriculture, human health, and societal sustainability.

Vanadate inhibition of fungal PhyA and bacterial AppA2 histidine acid phosphatases.

The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shut down by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal PhyA phytase and bacterial AppA2 phytase, kinetic experiments were performed in the presence and absence of orthovanadate and metavanadate under various acidic pHs. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 µM by orthovanadate. However, metavanadate preferentially inhibited the bacterial AppA2 phytase (50% inhibition at 8 µM) over the fungal phytase (50% inhibition at 40 µM). While in bacterial phytase the K(m) was not affected by ortho- or metavanadate, the V(max) was reduced. In fungal phytase, both the K(m) and V(max) was lowered. The vanadate exists as an anion at pH 3.0 and possibly binds to the active center of phytases that has a cluster of positively charged Arg, Lys, and His residues below the enzymes' isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serve as a molecular switch to turn off phytase activity while turning on the haloperoxidase activity. The fungal PhyA phytase's active site housing two distinct reactive centers, one for phosphomonoesterase and the other for haloperoxidase, is a unique example of how one protein could catalyze two dissimilar reactions controlled by vanadate.

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization.

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58 degrees C to 53 degrees C for bridge number 1, 37 degrees C for bridge number 3 and 4, and 42 degrees C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K (m) was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.

Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases.

Aspergillus niger PhyA and Escherichia coli AppA2 are increasingly used in animal feed for phosphorus nutrition and environmental protection. The objective of this study was to determine the impacts of assay conditions on activity estimates of these two phytases and to compare their biochemical characteristics at a pH similar to the stomach environment. The activities of the unpurified AppA2 were more variable than those of PhyA with three commonly used phytase activity assays. The variations associated with AppA2 were accounted for by buffer, pH, and the inclusion of Triton X-100 and BSA by approximately one-third each. At the commonly observed stomach pH of 3.5, the purified AppA2 had a lower affinity to phytate (a higher K(m)), but greater V(max), k(cat), and k(cat)/K(m) than those of PhyA. In summary, differences between AppA2 and PhyA in responses to activity assay conditions and in inherent kinetic properties should be considered in interpreting their feeding efficacy.

Unfolding and refolding of Aspergillus niger PhyB phytase: role of disulfide bridges.

The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.

Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases.

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The Km for phosphotyrosine ranged from 465 to 590 microM as opposed to 135 to 160 microM for phytate. The Vmax, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 x 10(6) to 1.6 x 10(7) M(-1) s(-1)); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5 degrees C higher to 70 degrees C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.

Salt effect on the pH profile and kinetic parameters of microbial phytases.

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.0. A high concentration of sodium chloride affected the activity of fungal phytase in the pH 3-4 range and shifted the pH optimum to 2.0 from 5.5 in Escherichia coli phytase. As expected, both of the microbial phytases were inhibited by sodium fluoride at acidic pH values. Because the Km for phytate increased nearly 2-fold for fungal phytase while Vmax increased about 75% in a high concentration of sodium chloride, it is possible that salt enhanced the product to dissociate from the active site due to an altered electrostatic environment. Modeling studies indicate that while the active site octapeptide's orientation is very similar, there are some differences in the arrangements of alpha-helices, beta-sheets, and coils that could account for the observed catalytic and salt effect differences.

Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive.

Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering.

Conservation of cysteine residues in fungal histidine acid phytases.

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering.

Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by guanidinium chloride and then refolded by removing the denaturant by dialysis. Thiol reagents prevented refolding; thus, disulfide bridge formation is an integral step in phytase folding. Catalytic demise of phytase after unfolding and refolding in presence of Tris(2-carboxyethyl)phosphine (TCEP) indicates that disulfide bridges are necessary for refolding. The hydrodynamic radius (rh) of active and unfolded phytase is 4 and 14 nm, respectively. Removal of denaturant through dialysis refolds phytase; its rh shifts back to 4 nm. When TCEP remains in the refolding media, the rh remains high. The unfolded phytase when diluted in assay medium refolds as a function of time at 25 and 37 degrees C, but not at higher temperature. Monitoring rh under denaturing and renaturing condition gives an accurate measure of the folding status of phytase.

Fungal phyA gene expressed in potato leaves produces active and stable phytase.

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 microM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.

PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases.

PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0.

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.

Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.