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? 2023 SAABL-myo-inositol-1-phosphate synthase (MIPS) and its product, free myo-inositol have been isolated from different bryophytes. MIPS was purified for the first time from the moss, Sphagnum junghuhnianum to 58.67 fold over i...
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? 2023 SAABL-myo-inositol-1-phosphate synthase (MIPS) and its product, free myo-inositol have been isolated from different bryophytes. MIPS was purified for the first time from the moss, Sphagnum junghuhnianum to 58.67 fold over its homogenate fraction with about 32.86 % recovery. D-glucose-6-phosphate was the exclusive substrate of the enzyme without which there was no enzyme activity and the deduction of NH4Cl, ME and NAD+ substantially reduced the activity of the enzyme. The Km for D-glucose-6-phosphate and NAD+were 1.81 mM and 0.25 mM, while the Vmax for the same were 1.42 mM and 1.12 mM respectively. The molecular weight of the enzyme was assessed to be approximately 174 kDa. The activity of the enzyme increased with the increase in the duration of incubation time for up to 90 min and with the increase in protein concentration for up to 300 μg. The pH and temperature maxima were pH 7.0 and at 30 °C, respectively, but a significant activity was observed at 10 °C also. NH4Cl substantially stimulated the enzyme activity while K+ and Ca2+ also raised the activity slightly. Li+ greatly inhibited the activity. Inhibitory activity was also shown by Cd2+, Mn2+, Zn2+and Hg2+ of which Hg2+ showed the maximum inhibition. Interestingly, the Sphagnum junghuhnianum MIPS showed the characteristics of both Class-II and Class-III aldolase.
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An efficient synthesis of tri-, tetra-, and pentasaccharide cyclic phosphates 1-5, structurally related to natural inositol phosphate glycans, is reported. The title compounds were assembled by PhSeOTf-promoted glycosylation of th...
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An efficient synthesis of tri-, tetra-, and pentasaccharide cyclic phosphates 1-5, structurally related to natural inositol phosphate glycans, is reported. The title compounds were assembled by PhSeOTf-promoted glycosylation of the known glucosamine precursor, t-butyldimethylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-#beta#-D-glucopyranoside (8) with protected 1-mehylthio mono-, di-, and trimannosides 7a-c, and, after conversion into glycosyl fluorides, Cp_2ZrCl_2-AgOTf-promoted glycosylation of differentially protected optically pure 1D-myo-inositol 11. The syntheses were completed by installing the cyclic phosphate moieties with methylpyridinium dichlorophosphate and finally, removal of all protecting groups by dissolving-metal reduction.
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L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate prec...
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L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0-60 % ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46 % of MIPS could be recovered. The human fetal liver MIPS specifically utilised D-glucose-6-phosphte and NAD ~+ as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40 °C. The enzyme activity was remarkably stimulated by NH4 ~+, slightly stimulated by K ~+ and Ca2+ and highly inhibited by Zn ~(2+), Cu ~(2+) and Hg ~(2+). The Km values of MIPS for D-glucose-6-phosphate and NAD ~+ were found to be as 1.15 and 0.12 mM respectively while the Vmax values were 280 nM and 252 nM for D-glucose-6- phosphate and NAD ~+ correspondingly. The apparent molecular weight of the native enzyme was determined to be 170 kDa.
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We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS). The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene. The putative acti...
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We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS). The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene. The putative active site residues are well conserved in Drosophila INOS protein. Southern blot analysis shows that Drosophila INOS gene is a single copy gene. Northern blot analysis reveals that Drosophila INOS gene expresses a 2.0-kb transcript that is more abundant in the head than the body, suggesting that it may be involved in brain function. The recombinant Drosophila INOS protein was expressed in Escherichia coli and the purified protein has proved to have a myo-inositol-1-phosphate synthase activity.
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The formation and degradation of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are of great metabolic importance, because of its role in the mediation of calcium release from intracellular stores. The conc...
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The formation and degradation of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are of great metabolic importance, because of its role in the mediation of calcium release from intracellular stores. The concentration of Ins(1,4,5)P3 in the cell is regulated by three signaling enzymes: phospholipase C isoforms release Ins(1,4,5)P3 from the plasma membrane by hydrolysis of phosphatidyl inositol 4,5-bisphosphate, whereas inositol phosphate 5-phosphatases remove it by dephosphorylation and a group of inositol phosphate kinases eliminate it by further phosphorylation at its 3- or 6-hydroxy group. The latter group is formed by the three isoforms of Ins(1,4,5)P3 3-kinase (IP3K) and inositol phosphate multikinase. In this article the tissue specific gene expression, molecular structure, role in calcium oscillations, regulation by calcium calmodulin, by phosphorylation and by intracellular localization of the IP3K isoforms are discussed. Another important aspect is the evolution of diverse inositol phosphate metabolizing enzymes from a eukaryotic founder by different mechanisms of gene diversification. Finally the role of IPMK in calcium signaling will be elucidated in more detail.
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The unique steps in the synthesis of an unusual osmolyte in hyperthermophiles, di-myo-inositol-1,1′-phosphate (DIP), involve the production of CDP-inositol and its condensation with an inositol-1-phosphate molecule to form phosph...
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The unique steps in the synthesis of an unusual osmolyte in hyperthermophiles, di-myo-inositol-1,1′-phosphate (DIP), involve the production of CDP-inositol and its condensation with an inositol-1-phosphate molecule to form phosphorylated DIP. While many organisms fuse both activities into a single enzyme, the two are separate in Thermotoga maritima. The crystal structure of the T. maritima inositol-1-phosphate cytidylyltransferase, which as a soluble protein may transiently associate with its membrane-embedded partner phospho-DIP synthase (P-DIPS), has now been obtained. The structure shows a conserved motif of sugar nucleotide transferases (COG1213) with a structurally reinforced C-terminal Cys bonded to the core of the protein. A bound arsenosugar identifies the location of the active site for inositol 1-phosphate. Based on homologous structures from several species and the identification of the crucial conserved aspartate residue, a catalytic mechanism for this enzyme is proposed as well as a mode for its association with P-DIPS. This structure imposes constraints on the mode of association, communication and temperature activation of two separate enzymes in T. maritima. For the first time, a working model for the membrane-bound P-DIPS unit has been constructed. This sheds light on the functioning of the phosphatidylserine and phosphatidylinositol synthases involved in many physiological processes that are homologous to P-DIPS. This work provides fresh insights into the synthesis of the unusual thermoprotective compound DIP in hyperthermophiles.
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From in vitro experiments with dialyzed cell-free extracts of the hyperthermophilic Archaeum Pyrococcus woesei, the biosynthetic pathway of di-myo-inositol-1,1'-phosphate was deduced. Starting from glucose-6-phosphate, the synthes...
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From in vitro experiments with dialyzed cell-free extracts of the hyperthermophilic Archaeum Pyrococcus woesei, the biosynthetic pathway of di-myo-inositol-1,1'-phosphate was deduced. Starting from glucose-6-phosphate, the synthesis proceeds in two steps with L-myo-inositol 1-phosphate as intermediate. (1) Interconversion of glucose 6-phosphate to inositol l-phosphate was observed without adding cofactors, such as nucleoside triphosphates or pyridine dinucleotides suggesting that the first enzyme reaction corresponds to the NAD(+)-dependent inositol 1-phosphate synthase reaction as already described for eukaryal systems, but differing from the latter by a stronger pyridine dinucleotide binding rendering the enzyme virtually independent from external NAD(+). (2) In a second step, two L-myo-inositol 1-phosphates are coupled under the expense of NTP to yield di-myo-inositol-1,1'-phosphate. The coupling of two L-myo-inositol 1-phosphates without preceding dephosphorylation of one of both by a phosphorylase as proposed for the di-myo-inositol-1,1'-phosphate synthesis in Methanococcus igneus (Chen, L. and Roberts, M. (1998) Appl. Environ. Microbiol. 64, 2609-2615) is supported by labeling experiments which resulted only in a labeled product with L-myo [U-14C]inositol 1-phosphate, but not with radiolabeled L-myo-[U-C-14]inositol and non-labeled L-myo-inositol 1-phosphate. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. [References: 12]
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Phospholipases D (PLDs), the major dermonecrotic factors from brown spider venoms, trigger a range of biological reactions both in vitro and in vivo. Despite their clinical relevance in loxoscelism, structural data is restricted t...
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Phospholipases D (PLDs), the major dermonecrotic factors from brown spider venoms, trigger a range of biological reactions both in vitro and in vivo. Despite their clinical relevance in loxoscelism, structural data is restricted to the apo-form of these enzymes, which has been instrumental in understanding the functional differences between the class I and II spider PLDs. The crystal structures of the native class II PLD from Loxosceles intermedia complexed with myo-inositol 1-phosphate and the inactive mutant H12A complexed with fatty acids indicate the existence of a strong ligand-dependent conformation change of the highly conserved aromatic residues, Tyr 223 and Trp225 indicating their roles in substrate binding. These results provided insights into the structural determinants for substrate recognition and binding by class II PLDs.
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Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecul...
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Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay - the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg~(2+) and is not enhanced by other divalent metal ions (Zn~(2+) and Mn ~(2+)), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.
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An integrated process was developed to hydrolyze the phytates in light steep water (LSW) and to simultaneously isolate inorganic phosphate (Pi) and myo-inositol products. The proposed integrated process will be helpful in resolvin...
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An integrated process was developed to hydrolyze the phytates in light steep water (LSW) and to simultaneously isolate inorganic phosphate (Pi) and myo-inositol products. The proposed integrated process will be helpful in resolving the environmental and nutritional concerns in the use of corn gluten feed (CGF) in the animal diets. This process comprised of partial and total hydrolysis of LSW and intermediate anion exchange separation technique. The phytates in LSW were initially degraded to negatively charged myo-inositol phosphates (InsP_2-InsP_5). The optimized experimental parameters for the partial hydrolysis of LSW were determined to be 2h hydrolysis with 1FTU Aspergillus niger/g substrate at 35°C. The negatively charged species of the partially hydrolyzed substrate were separated on a strong base anion exchange resin. The negatively charged species, retained by the resin, were eluded with 1M NaCl solution and were subjected to complete hydrolysis with the Escherichia coli, A. niger derived phytases and their respective combinations. The maximum amount of myo-inositol released from the anion exchange column was 3.73±0.03mg/NaCl elution which was detected after 48h reactions catalyzed by 100FTU E. coli, 150FTU E. coli, and 150FTU the combination of A. niger and E. coli. The time course of Pi released showed a similar trend to that of myo-inositol and the released Pi reached a maximum amount of 3.30±0.05mg/g NaCl elution after 48h incubation at the enzyme loadings for which the maximum concentration of myo-inositol were reached.
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