Biochemical properties and substrate specificities of alkaline and histidine acid phytases

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg⁻¹ protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg⁻¹ of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Molecular characterization,physicochemical properties,known and potential applications of phytases: An overview

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze the phosphate ester bonds of phytate-releasing phosphate and lower myo-inositol phosphates and/or myo-inositol. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly. In this study, the molecular, biophysical, and biochemical properties of phytases are reviewed in detail. Alterations in the molecular and catalytic properties of phytases, upon expression in heterologous hosts, are discussed. Diverse applications of phytases as feed additives, as soil amendment, in aquaculture, development of transgenic organisms, and as nutraceuticals in the human diet also are dealt with. Furthermore, phytases are envisaged to serve as potential enzymes that can produce versatile lower myo-inositol phosphates of pharmaceutical importance. Development of phytases with improved attributes is an important area being explored through genetic and protein engineering approaches, as no known phytase can fulfill all the properties of an ideal feed additive.     

In <Emphasis Type="Italic">silico</Emphasis> study on the substrate binding manner in human <Emphasis Type="Italic">myo</Emphasis>-inositol monophosphatase 2

The human IMPA2 gene encoding myo-inositol monophosphatase 2 is highly implicated with bipolar disorder but the substrates and the reaction mechanism of myo-inositol monophosphatase 2 have not been well elucidated.9 In the present study, we constructed 3D models of three- and two-Mg²⁺-ion bound myo-inositol monophosphatase 2, and studied substrate-binding manners using the docking program AutoDock3. The subsequent study showed that the three-metal-ion model could interact with myo-inositol monophosphates, as follows: The phosphate moiety coordinated three Mg²⁺ ions, and the inositol ring formed hydrogen bonds with the amino acids conserved in the family. Furthermore, the OH group vicinal to the phosphate group formed a hydrogen bond with a non-bridging oxygen atom of the phosphate. These interactions have been proposed as crucial for forming the transitional state, bipyramidal structure in the bovine myo-inositol monophosphatase. We therefore propose that the human myo-inositol monophosphatase 2 interacts with myo-inositol monophosphates in the three-metal-ion bound form, and proceeds the dephosphorylation through the three-metal-ion theory.     

An examination of some possible sources of soil inositol phosphates

Summary Examination of extracts prepared from soil fungi and yeast cells (S. cavlsbergensis) showed that phytate phosphorus was not present. Phytin was successfully extracted from the acorns ofQuercus robur andQ. hodgkinsonii but was apparently absent from acorns ofQ. ilex. Acorn phytin was shown to consist only of phosphate esters ofmyo-inositol although freescyllo-inositol is present in this plant tissue. Small amounts of phytate phosphorus were isolated from sand/clay cultures, after a period of incubation, but onlymyo-inositol derivatives appeared to be present.     

Phenotypic,genetic and molecular characterization of a maize low phytic acid mutant (lpa241)

Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the major storage compound of phosphorous (P) in plants, predominantly accumulating in seeds (up to 4–5% of dry weight) and pollen. In cereals, phytic acid is deposited in embryo and aleurone grain tissues as a mixed "phytate" salt of potassium and magnesium, although phytates contain other mineral cations such as iron and zinc. During germination, phytates are broken down by the action of phytases, releasing their P, minerals and myo-inositol which become available to the growing seedling. Phytic acid represents an anti-nutritional factor for animals, and isolation of maize low phytic acid (lpa) mutants provides a novel approach to study its biochemical pathway and to tackle the nutritional problems associated with it. Following chemical mutagenesis of pollen, we have isolated a viable recessive mutant named lpa 241 showing about 90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway.Communicated by F. Salamini     

Dietary phytase and myo-inositol supplementation are associated with distinct plasma metabolome profile in broiler chickens

Phytase enzyme is used as a dietary supplement in broiler nutrition to improve phosphorous bioavailability. Phytase deliberates phosphate groups from phytic acid and produces myo-inositol after total dephosphorylation. Myo-inositol is a bioactive compound having beneficial modulatory effects on metabolism in humans. However, it is not well understood if and how phytic acid degradation products, particularly myo-inositol, can modulate metabolism in broiler chicken. The purpose of this study was to investigate effects of dietary supplements of phytase and myo-inositol on the blood plasma metabolome profile of broiler chickens. Broilers were provided a nutrient-adequate control diet or the same diet supplemented with either 3.5 g myo-inositol or 500, 1500 or 3000 units of phytase, per kilogram of feed (grower diet). Broilers were group-housed in floor pens (eight pens per diet) and provided one of the treatment diets for 22 days. Then, blood was collected from one bird per pen, resulting in eight replicated measurements per diet. A targeted metabolomics approach was applied to the heparin plasma. Body weight of the birds was not significantly affected by the treatments. Plasma myo-inositol concentrations were significantly increased by myo-inositol supplementation and phytase supplementation at 500 and 1500 units/kg. Metabolites generally affected by phytase supplementation belonged to the groups of acyl-carnitines, phosphatidylcholines, sphingomyelins, lysophosphatidylcholine, biogenic amines and amino acids. Compared to the control diet, phytase supplements had significantly higher plasma concentrations of kynurenine and creatinine, but lower concentrations of histamine and cis-4-hydroxyproline. Myo-inositol supplementation significantly increased plasma concentrations of dopamine and serotonine. While some metabolites were similarly affected by myo-inositol and phytase supplementation, others were distinctly differently affected. We conclude that myo-inositol, either as a directly added supplement or indirectly released from phytate upon phytase supplementation, can affect specific metabolic pathways. Additional effects found on phytase supplementation may be related to intermediary phytate degradation products. Results are indicative for innovative hypothesis to be tested in future experiments, for instance, with regard to relationships between phytase or myo-inositol supplements and bird immunity or behaviour.     

Inheritance of seed phytate and phosphorus levels in common bean (Phaseolus vulgaris L.) and association with newly-mapped candidate genes

Common bean (Phaseolus vulgaris L.) is an important, high-quality staple food that provides large amounts of protein and mineral micronutrients to the diets of people in many countries. Phytates are a storage form of organic phosphorus which is used by the plant in various stages of growth and development but can have certain anti-nutrient properties due to chelation of minerals such as iron and zinc. At the same time, phytates provide certain health benefits and therefore are the subject of both mutagenesis and breeding programs for functional foods. The objective of this study was to evaluate the quantitative trait loci (QTL) associated with seed phytate and seed phosphorus concentration and content on a per-seed basis and to develop functional molecular markers for genes from the phytic acid synthesis pathway. We used a well-characterized mapping population, DOR364?×?G19833, in three field experiments with three repetitions each and two levels of soil phosphorus fertilization, as well as a large set of previously and newly developed primer pairs for the genes myo-inositol (3)P₁ synthase, myo-inositol kinase and various inositol kinases. We identified an association of phytate concentration QTL with one of two paralogs of the myo-inositol (3)P₁ synthase gene family, located on linkage group b01 and expressed in common bean seed rather than in vegetative tissues. We also identified QTL for phytate concentration on linkage group b06 and phytate content on linkage groups b03, b04 and b10. We provide a synteny analysis based on common bean versus soybean genome comparisons of all the phytic acid pathway genes that were genetically mapped and indicate flanking markers that can be used for marker-assisted selection when the genes themselves are not polymorphic as PCR amplicons. We can conclude that natural variability in phytate levels is controlled by the seed-expressed myo-inositol (3)P₁ synthase gene (MIPS) as well as other loci in the common bean genome. This means that breeding of phytate levels in common bean must take into account allele variability at certain candidate genes, such as this seed MIPS gene, a recently cloned ABC trasnporter and additional QTL for the trait, which underlie the oligogenic inheritance for phytate concentration in common bean.     

The enzymes involved in the synthesis of phytic acid in Lemna gibba (Studies on the biosynthesis of cyclitols,XL.)

Summary The biosynthesis of phytic acid is known to be catalyzed by enzymes causing a stepwise phosphorylation of myo-inositol or 1l-myo-inositol 1-phosphate with adenosine triphosphate as phosphate donor. The kinases responsible for these phosphorylations in Lemna gibba were purified by affinity chromatography on a Sepharose gel carrying myo-inositol 2-phosphate at the binding site. Three fractions with enzymatic activity could be identified; in the first one, we find myo-inositol kinase (EC 2.7.1.64) phosphorylating myo-inositol to 1l-myo-inositol 1-phosphate; the second one brings about the phosphorylation of myo-inositol trisphosphate to phytic acid; the third one phosphorylates myo-inositol 1-phosphate to a myo-inositol trisphosphate. An enzyme oxidizing 1l-myo-inositol 1-phosphate to an uronic acid derivative is found in the first two fractions. In the presence of ATP, Mg²⁺ Mn²⁺, and the second and the third enzyme fractions in an appropriate mixture, 1l-myo-inositol 1-phosphate can be phosphorylated to phytic acid. The structure of the trisphosphate acting as an intermediate is not yet known.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Complete hydrolysis of <Emphasis Type="Italic">myo</Emphasis>-inositol hexakisphosphate by a novel phytase from <Emphasis Type="Italic">Debaryomyces castellii</Emphasis> CBS 2923

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K _m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P₆) to inositol and inorganic phosphate. The sequence of Ins P₆ hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P₅, Ins (1,2,5,6) P₄, Ins (1,2,6) P₃, Ins (1,2) P₂, Ins (1 or 2) P₁, and inositol (notation 3/4/5/6/1 or 2).     

<Emphasis Type="Italic">myo</Emphasis>-Inositol Phosphate Isomers Generated by the Action of a Phytase from a Malaysian Waste-water Bacterium

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P₅, D-I(2,3,4,5)P₄, D-I(2,3,4)P₃, D-I(2,3)P₂ to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P₅. Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P₅, D/L-I(1,2,4,5)P₄, D/L-I(1,2,4)P₃, D/L-I(2,4)P₂ to finally I(2)P.     

Partial purification and characterization of phytases from pollen of lily (Lilium longiflorum Thunb.)

Summary Two phytases from lily pollen (Lilium longiflorum Thunb.) were partially purified and characterized. The first (pH optimum 5.0) was purified 40-fold from ungerminated pollen. The second (pH optimum 6.5) appeared during germination and was purified 68-fold from pollen germinated 2 h. Molecular weight of the first was 72 kD, and the second was 36 kD as determined by gel filtration. Both were active against phosphate esters other than phytate, although purification of the first reduced its activity against AMP and myo-inositol 2-P to 10% of activity against phytate. Phytase from germinated pollen (but not ungerminated) was inhibited by the sulfhydryl agent parahydroxy mercuribenzoate; P _i inhibited phytase from ungerminated but not germinated pollen. Such different catalytic and physical properties may reflect different biochemical functions.Abbreviations HPLC High performance liquid chromatography - DEAE diethyl aminoethyl - P _i orthophosphate - PP _i pyrophosphate - p-NPP para-nitrophenyl phosphate - pNP para-nitrophenol - MI myo-inositol - MI 2-P myo-inositol 2-P - MI penta P myo-inositol pentakisphosphate - PHMB para-hydroxy mercuribenzoate - PMSF phenyl methyl sulfonyl fluoride - AMP adenosine monophosphate - GMP guanosine monophosphate - EGTA ethylene glycol-bis (-aminoethyl ether) N, N, N, N-tetraacetic acid     

Down-regulation of the <Emphasis Type="Italic">myo</Emphasis>-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis

The enzyme myo-inositol oxygenase (MIOX; E.C. 1.13.99.1) catalyzes the ring-opening four-electron oxidation of myo-inositol into glucuronic acid, which is subsequently activated to UDP-glucuronic acid (UDP-GlcA) and serves as a precursor for plant cell wall polysaccharides. Starting from single T-DNA insertion lines in different MIOX-genes a quadruple knockdown (miox1/2/4/5-mutant) was obtained by crossing, which exhibits greater than 90% down-regulation of all four functional MIOX genes. Miox1/2/4/5-mutant shows no visible phenotype and produces viable pollen. The alternative pathway to UDP-glucuronic acid via UDP-glucose is upregulated in the miox1/2/4/5-mutant as a compensatory mechanism. Miox1/2/4/5-mutant is impaired in the utilization of myo-inositol for seedling growth. The incorporation of myo-inositol derived sugars into cell walls is strongly (>90%) inhibited. Instead, myo-inositol and metabolites produced from myo-inositol such as galactinol accumulate in the miox1/2/4/5-mutant. The increase in galactinol and raffinose family oligosaccharides does not enhance stress tolerance. The ascorbic acid levels are the same in mutant and wild type plants.     

Ice-nucleating activity of Pseudomonas syringae cultivated on a natural substrate; influence of phosphate

A novel kind of culture medium is used for the production Pseudomonas syringae with a high ice-nucleating activity. It is based on a natural substrate, wheat bran, which contains a relatively high proportion of phytate. The double salt of phytic acid is the precursor of a major component of the ice-nucleating site (myo-inositol). Experiments with the purified components show the positive effects on the ice-nucleating activity. The use of the wheat bran medium seems to be specifically efficient on class A bacteria, which is the most active type of P. syringae. We have shown that inorganic phosphate starvation during the preculture of P. syringae leads to higher ice-nucleating activity.     

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis α-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the δ-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and α-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.     

Digalactosyldiacylglycerols Isolated from a Brown Alga as Effective Phagostimulants for a Young Abalone

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His₆-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.