Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg²⁺ together suggests sulfhydryl involvement at the active site.     

Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase

Chloroplasts from 5 to 7 day old Vigna radiata seedling, grown under alternate light/dark conditions or from green Euglena gracilis Z. cells have been found to harbor L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) activity. In contrast, dark-grown V. radiata seedlings, or streptomycin-bleached Euglena cells exhibit either reduced or no enzyme activity. An apparent enhancement of the chloroplastic inositol synthase by growth in presence of light is observed.     

myo-Inositol-1-phosphate Synthase Is Required for Polar Auxin Transport and Organ Development

myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all null mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution.     

myo-Inositol metabolism in rat testis in response to streptozotocin-induced diabetes

The effects of streptozotocin-induced hyperglycemia on de novo myo-inositol biosynthesis in rat testis was examined. Testicular glucose and glucose 6-phosphate levels increased significantly 10 and 12 h after stretozotocin injection, respectively. However, testis myo-inositol content did not increase appreciably until 24 h following injection of the drug. Seventy-two hours after streptozotocin administration, testis myo-inositol levels were 2.7-fold higher in diabetic rats than in controls injected with citrate buffer. No changes were observed in the Specific activities of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and 1-l-myo-inositol-1-phosphatase (EC 3.1.3.25). However, hyperglycemic rats displayed testicular glucose and glucose 6-phosphate levels approximately 4- and 2-fold in excess of control values, respectively. Insulin treatment of diabetic rats resulted in the lowering of plasma glucose, and testis glucose 6-phosphate to normal or below normal levels within hours. Inositol levels remained significantly elevated compared with control animals, although slightly lower than that observed for untreated diabetic rats. Streptozotocin diabetic rats had a significantly decreased testis cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio compared with control animals 72 h after injection. The potential role of testis hexokinase distribution in the regulation of glucose 6-phosphate and myo-inositol biosynthesis in normal and diabetic rats was investigated. No significant differences in testis hexokinase distribution or in the kinetic characteristics of the soluble and particulate hexokinase activities were observed. Testicular sperm counts in streptozotocin diabetic rats were not significantly different from control values.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

Myo-inositol-1-phosphate synthase from streptomyces griseus (Studies on the biosynthesis of cyclitols,XXXVIII1)

Summary It could be shown that Streptomyces griseus, the microorganism producing the antibiotic streptomycin and also mutant strains of this species that cannot synthesize streptomycin, possess myo-inositol-1-phosphate synthase (EC 5.5.1.4), the enzyme cyclizing D-glucose 6-phosphate. The enzyme isolated from that organism is extremely instable, its molecular weight is approximately 260,000, and it requires a divalent metal ion for its activity. This is the first instance that an enzyme of this specificity has been found in a prokaryotic organism.     

Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme.     

Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat

The myo-inositol level of plasma was determined during pre- and postnatal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly 10-fold. Immediately after birth, the myo-inositol level decreased but was maintained at values 3–4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 mM.The biosynthesis of myo-inositol from glucose 6-phosphate is catalyzed by glucose 6-phosphate:l-myo-inositol-1-phosphate cyclase and l-myo-inositol-1-phosphate phosphatase. The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that the fetal liver accounted for over 48% of the total carcass cyclase and 26% of the total carcass phosphatase activity. Developmental changes correlated well with the pattern of myo-inositol in fetal rat plasma. Similarly, the enzymes of the myo-inositol biosynthetic pathway increased in rat mammary gland in close agreement with the myo-inositol content of milk and diminished to prelactation activities within 24 hr after the onset of involution.The myo-inositol level of colostrum and milk of five human subjects was highest (2.8 mM) before birth and decreased to 40% of that level 5 days postpartum, where it remained for at least 3 weeks. Even after 7 months of lactation, the milk of one subject contained 3–4-fold more myo-inositol than all commercial infant formulas analyzed.     

myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d-glucuronic Acid Biosynthesis in Plants

Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all.     

Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus spp.

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, α-glutamate, di-myo-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated α-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.     

Chemical synthesis of diphosphoinositide

A chemical synthesis of DL-1-O-(1′-palmitoyl-2′-oleoyl-sn-glycero-3′-phosphoryl)-myo-inositol-4-phosphate (diphosphoinositide) is described. Selective phosphorylation of DL-2,3; 5,6-di-O-cyclohexylidene-myo-inositol with diphenylphosphochloridate led to the corresponding 1-diphenylphosphate which was transformed into silver DL-4-O-acetyl-2,3; 5,6-di-O-cyclohexylidene-myo-inositol-1-(benzyl)phosphate. Condensation of the latter with 1-palmitoyl-2-oleoyl-sn-glycero-3-iodohydrin gave a phosphotriester which after successive deacetylation, phosphorus oxychloride treatment and removal of the protective groups yielded diphosphoinositide. The intermediate DL-1-O-[1′-palmitoyl-2′-oleoyl-sn-glycero-3′-(benzyl)phosphoryl]-2,3; 5,6-di-O-cyclohexylidene-myo-inositol was used also for a new synthesis of phosphatidylinositol.     

Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.     

The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme’s preference for the 3′-phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced-fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability.     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.     

Biosynthesis of myo-inositol in rat mammary gland. Isolation and properties of the enzymes

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) and 1l-myo-inositol 1-phosphatase (EC 3.1.3.25) were isolated and partially purified from lactating rat mammary gland. The synthase had an apparent molecular weight of 290,000 as determined by gel filtration; its pH optimum was 7.2, and the K_m for glucose 6-phosphate was 0.5 mm. No other compound could act as a substrate, but the synthase was inhibited 100% by d-gluconic acid 6-phosphate, 54% by d-fructose 6-phosphate, 31.8% by d-galactose 6-phosphate, and 29.6% by d-mannose 6-phosphate each at 5mm. Activity was stimulated 2-fold by the addition of 1 mm NAD⁺ and 40% by 14 mm ammonium ions, whereas it was inhibited by 30% in the presence of 1 mm NADH and by 93.6% when incubated with 1 mmp-mercuribenzoate. Reagents which interfere with Schiff-base formation, pyridoxal 5′-phosphate and trinitrobenzenesulfonate, inhibited the enzyme, but EDTA was without effect.The 1l-myo-inositol 1-phosphatase from rat mammary tissue appears to exist in a native tetrameric form of 210,000 as determined by gel filtration which, upon heating at 70 °C for 15 min, is converted into a stable monomer of approximately 52,000. Mg²⁺ (1.5 mm) was an absolute requirement for activity though Mn²⁺ gave 17% of the activity provided by Mg²⁺. Sodium, potassium, or ammonium ions were stimulatory, but lithium ions were strongly inhibitory. 1l-myo-Inositol 1-phosphatase specifically cleaved 1l-myo-inositol 1-phosphate and was 60% as active toward l-α-glycerol phosphate with only minor activity toward other phosphorylated compounds. The pH optimum was 8.0 and the K_m for 1l-myo-inositol 1-phosphate was 0.8 mm.     

A Novel Biosynthetic Pathway of Archaetidyl-myo-inositol via Archaetidyl-myo-inositol Phosphate from CDP-archaeol and d-Glucose 6-Phosphate in Methanoarchaeon Methanothermobacter thermautotrophicus Cells

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[¹⁴C]inositol 1-phosphate, which was enzymatically prepared from [¹⁴C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )     

Studies on avian erythrocyte metabolism: purification and properties of myo-inositol 1-phosphatase form chick erythrocytes

An enzyme capable of hydrolyzing myo-inositol 1-phosphate was identified and partially purified from the erythrocytes of 7-day chicks. It has an apparent molecular weight of approximately 60,000, is heat stable, and has a pH of optimal activity between 6.5 and 7.3. In most regards the kinetic properties are similar to the myo-inositol 1-phosphatases of rat testis, rat mammary gland, bovine brain, and of yeast. The enzyme has an absolute requirement for a divalent cation; Mg²⁺ gave the greatest activity, with an optimal concentration of 2.5 mm in the standard assay employed. Zn²⁺, Co²⁺, and Mn²⁺ supported activity to a lesser degree. Activity was inhibited by NaF, HgCl₂, and p-hydroxymercuribenzoate. myo-Inositol tetrakis (dihydrogen phosphate) and myo-inositol 1,3,4,5,6-pentakis (dihydrogen phosphate) were not substrates for this enzyme and inhibited the hydrolysis of myo-inositol 1-phosphate. Unlike other phosphatases for myo-inositol 1-phosphate, this enzyme cleaved myo-inositol 1-phosphate (K_m = 8.6 × 10^?5 m) and myo-inositol 2-phosphate (K_m = 2.86 × 10^?4 m) at approximately the same rates. It also hydrolyzed 2′-purine and pyrimidine ribonucleotides about as well as myo-inositol 1-phosphate, but was only 20–30% as active against the 3′-ribonucleotides and had scarcely any activity against the 5′-ribonucleotides. The amount of enzyme activity in erythrocytes of embryos, chicks, and mature chickens was the same (~29 μmol/ml rbc/h). The biological function of this enzyme in avian erythrocytes is unclear at this time. Other tissues containing this phosphatase also have an enzyme which synthesizes myo-inositol 1-phosphate from glucose 6-phosphate, but we have been unable to detect the presence of such an enzyme in avian erythrocytes.