Biosynthesis Of Myo-inositol Research Articles

The host and pathogen myo-inositol-1-phosphate synthases are required for Rhizoctonia solani AG1-IA infection in tomato.

The myo-inositol-1-phosphate synthase (MIPS) catalyses the biosynthesis of myo-inositol, an important sugar that regulates various physiological and biochemical processes in plants. Here, we provide evidence that host (SlMIPS1) and pathogen (Rs_MIPS) myo-inositol-1-phosphate synthase (MIPS) genes are required for successful infection of Rhizoctonia solani, a devastating necrotrophic fungal pathogen, in tomato. Silencing of either SlMIPS1 or Rs_MIPS prevented disease, whereas an exogenous spray of myo-inositol enhanced disease severity. SlMIPS1 was upregulated upon R. solani infection, and potentially promoted source-to-sink transition, induced SWEET gene expression, and facilitated sugar availability in the infected tissues. In addition, salicylic acid (SA)-jasmonic acid homeostasis was altered and SA-mediated defence was suppressed; therefore, disease was promoted. On the other hand, silencing of SlMIPS1 limited sugar availability and induced SA-mediated defence to prevent R. solani infection. Virus-induced gene silencing of NPR1, a key gene in SA signalling, rendered SlMIPS1-silenced tomato lines susceptible to infection. These analyses suggest that induction of SA-mediated defence imparts disease tolerance in SlMIPS1-silenced tomato lines. In addition, we present evidence that SlMIPS1 and SA negatively regulate each other to modulate the defence response. SA treatment reduced SlMIPS1 expression and myo-inositol content in tomato, whereas myo-inositol treatment prevented SA-mediated defence. We emphasize that downregulation of host/pathogen MIPS can be an important strategy for controlling diseases caused by R. solani in agriculturally important crops.

Phosphonate and α-Fluorophosphonate Analogues of d-Glucose 6-Phosphate as Active-Site Probes of 1l-myo-Inositol 1-Phosphate Synthase.

The biosynthesis of myo-inositol (mI) is central to the function of many organisms across all kingdoms of life. The first and rate-limiting step in this pathway is catalyzed by 1l-myo-inositol 1-phosphate synthase (mIPS), which converts d-glucose 6-phosphate (G6P) into 1l-myo-inositol 1-phosphate (mI1P). Extensive studies have shown that this reaction occurs through a stepwise NAD+-dependent redox aldol cyclization mechanism producing enantiomerically pure mI1P. Although the stereochemical nature of the mechanism has been elucidated, there is a lack of understanding of the importance of amino acid residues in the active site. Crystal structures of mIPS in the ternary complex with substrate analogues and NAD(H) show different ligand orientations. We therefore proposed to use isosteric and isoelectronic analogues of G6P to probe the active site. Here, we report the synthesis of the methylenephosphonate, difluoromethylenephosphonate, and (R)- and (S)-monofluoromethylenephosphonate analogues of G6P and their evaluation as inhibitors of mIPS activity. While the CH2 and CF2 analogues were produced with slight modification of a previously described route, the CHF analogues were synthesized through a new, shorter pathway. Kinetic behavior shows that all compounds are reversible competitive inhibitors with respect to G6P, with Ki values in the order CF2 (0.18 mM) < (S)-CHF (0.24 mM) < (R)-CHF (0.59 mM) < CH2 (1.2 mM). Docking studies of these phosphonates using published crystal structures show that substitution of the oxygen atom of the substrate changes the conformation of the resulting inhibitors, altering the position of carbon-6 and carbon-5, and this change is more pronounced with fluorine substitution.

Potential of engineering the myo-inositol oxidation pathway to increase stress resilience in plants.

Myo-inositol is one of the most abundant form of inositol. The myo-inositol (MI) serves as substrate to diverse biosynthesis pathways and hence it is conserved across life forms. The biosynthesis of MI is well studied in animals. Beyond biosynthesis pathway, implications of MI pathway and enzymes hold potential implications in plant physiology and crop improvement. Myo-inositol oxygenase (MIOX) enzyme catabolize MI into D-glucuronic acid (D-GlcUA). The MIOX enzyme family is well studied across few plants. More recently, the MI associated pathway's crosstalk with other important biosynthesis and stress responsive pathways in plants has drawn attention. The overall outcome from different plant species studied so far are very suggestive that MI derivatives and associated pathways could open new directions to explore stress responsive novel metabolic networks. There are evidences for upregulation of MI metabolic pathway genes, specially MIOX under different stress condition. We also found MIOX genes getting differentially expressed according to developmental and stress signals in Arabidopsis and wheat. In this review we try to highlight the missing links and put forward a tailored view over myo-inositol oxidation pathway and MIOX proteins.

Control of fruit softening and Ascorbic acid accumulation by manipulation of SlIMP3 in tomato.

SummaryPostharvest deterioration is among the major challenges for the fruit industry. Regulation of the fruit softening rate is an effective strategy for extending shelf‐life and reducing the economic losses due postharvest deterioration. The tomato myoinositol monophosphatase 3 gene SlIMP3, which showed highest expression level in fruit, was expressed and purified. SlIMP3 demonstrated high affinity with the L‐Gal 1‐P and D‐Ins 3‐P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol. Overexpression of SlIMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, delayed fruit softening, decreased water loss, and extended shelf‐life. Overexpression of SlIMP3 also increased uronic acid, rhamnose, xylose, mannose, and galactose content in cell wall of fruit. Treating fruit with myoinositol obtained similar fruit phenotypes of SlIMP3‐overexpressed fruit, with increased cell wall thickness and delayed fruit softening. Meanwhile, overexpression of SlIMP3 conferred tomato fruit tolerance to Botrytis cinerea. The function of SlIMP3 in cell wall biogenesis and fruit softening were also verified using another tomato species, Ailsa Craig (AC). Overexpression of SlDHAR in fruit increased AsA content, but did not affect the cell wall thickness or fruit firmness and softening. The results support a critical role for SlIMP3 in AsA biosynthesis and cell wall biogenesis, and provide a new method of delaying tomato fruit softening, and insight into the link between AsA and cell wall metabolism.

SLC5A3-Dependent Myo-inositol Auxotrophy in Acute Myeloid Leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate-limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme. SIGNIFICANCE: We show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically defined subset of AML.This article is highlighted in the In This Issue feature, p. 275.

Biosynthesis of myo-inositol in Escherichia coli by engineering myo-inositol-1-phosphate pathway

Myo-inositol is widely used in drug, food and animal feed industries. In this study, the myo-inositol biosynthetic pathway was established in Escherichia coli by introducing the myo-inositol-1-phosphate synthase-encoding gene (INO1) from Saccharomyces cerevisiae SC288. The production of myo-inositol was also investigated in shake flasks. The results showed that gene INO1 was succefully expressed in E. coli as verified by SDS-PAGE analysis. The initial glucose concentrations in M9 media had a positive impact on the production of myo-inositol and acetic acid, OD600 of cells and plasmid stability. The highest concentration of myo-inositol was obtained at the initial glucose concentration of 30 g/L, which reached 887 mg/L. When the initial concentration of glucose was 40 g/L, a lot of acetic acid was accumulated (8.53 g/L), causing slightly decrease in the concentration of myo-inositol. The recombinant plasmid was more unstable at higher initial concentration of glucose as compared to a lower one. 797 mg/L of myo-inositol was ultimately produced in a 1 L shake flask at the initial glucose concentration of 10 g/L. The maximal OD600 of cells and the highest concentration of acetic acid were 7.18 and 3.05 g/L, respectively. This study lays good foundation for understanding the biosynthesis of myo-inositol in E. coli and provides the possibility of applying myo-inositol to the industrial production by recombinant E. coli in future.

The Function of Inositol Phosphatases in Plant Tolerance to Abiotic Stress

Inositol signaling is believed to play a crucial role in various aspects of plant growth and adaptation. As an important component in biosynthesis and degradation of myo-inositol and its derivatives, inositol phosphatases could hydrolyze the phosphate of the inositol ring, thus affecting inositol signaling. Until now, more than 30 members of inositol phosphatases have been identified in plants, which are classified intofive families, including inositol polyphosphate 5-phosphatases (5PTases), suppressor of actin (SAC) phosphatases, SAL1 phosphatases, inositol monophosphatase (IMP), and phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-related phosphatases. The current knowledge was revised here in relation to their substrates and function in response to abiotic stress. The potential mechanisms were also concluded with the focus on their activities of inositol phosphatases. The general working model might be that inositol phosphatases would degrade the Ins(1,4,5)P3 or phosphoinositides, subsequently resulting in altering Ca2+ release, abscisic acid (ABA) signaling, vesicle trafficking or other cellular processes.

MP88-07 THE ISYNA1 EXPRESSION IN PRIMARY RENAL CANCER AS A POTENTIAL PROGNOSTIC BIOMARKER FOR THE POST-OPERATIVE METASTASIS.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2018MP88-07 THE ISYNA1 EXPRESSION IN PRIMARY RENAL CANCER AS A POTENTIAL PROGNOSTIC BIOMARKER FOR THE POST-OPERATIVE METASTASIS. Tomoyuki Koguchi, Kei Ishibashi, Akifumi Onagi, Ryo Tanji, Ruriko Takinami, Seiji Hoshi, Junya Hata, Yuichi Sato, Hidenori Akaihata, Masao Kataoka, Soichiro Ogawa, Nobuhiro Haga, Ken Aikawa, and Yoshiyuki Kojima Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Kei IshibashiKei Ishibashi More articles by this author , Akifumi OnagiAkifumi Onagi More articles by this author , Ryo TanjiRyo Tanji More articles by this author , Ruriko TakinamiRuriko Takinami More articles by this author , Seiji HoshiSeiji Hoshi More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Hidenori AkaihataHidenori Akaihata More articles by this author , Masao KataokaMasao Kataoka More articles by this author , Soichiro OgawaSoichiro Ogawa More articles by this author , Nobuhiro HagaNobuhiro Haga More articles by this author , Ken AikawaKen Aikawa More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2927AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In response to cellular stress, p53 exerts its tumor suppressive effects such as apoptosis, cell cycle arrest, and cellular homeostasis through the induction of its target genes. On the other hand, Various tumors had p53 mutations, nevertheless renal cell carcinoma(RCC) had few p53 mutations. Previous study reported that p53 inactivated pathways affected the renal carcinogenesis. Other report showed that p53 expression pattern in immunohistochemistry analysis of tumor was associated prognosis of RCC. Therefore, we considered a possibility that p53 target gene might affect prognosis of RCC. We identified a novel p53 function, and examined whether the p53 target was useful as a prognostic factor of RCC. METHODS To screen novel p53 target genes, we conducted cDNA microarray analysis using mRNAs isolated from HCT116 p53+/+ and HCT116 p53-/- cells that were treated with 2 μg/ml of adriamycin (ADR). To investigate whether mRNA transcription was regulated by p53, we performed a reporter assay and a chromatin immunoprecipitation (ChIP) assay using U373MG cells. To evaluate the biosynthesis of myo-inositol by a novel p53 target, we performed myo-inositol (MI) assay using HEK293T cells that were transfected with plasmid expressing the p53 target, and using HCT116 p53+/+ and HCT116 p53-/- cells that were treated with ADR. To examined whether it was useful as a prognosis of RCC, we performed the overall survival (OS) analysis and progression free survival (PFS) analysis using mRNA expression of the p53 target in primary RCC. RESULTS The result of cDNA microarray analysis indicated ISYNA1 as a novel candidate gene. We found p53 response elements in the promoter region and the seventh exon by results of reporter assay and ChIP assay. Therefore, we identified ISYNA1 as a novel p53 target. The results of MI assay showed that intracellular myo-inositol content in cells expressing ISYNA1. In addition, DNA damage significantly increased intracellular myo-inositol content in HCT116 p53+/+ cells, but did not affect the myo-inositol content in HCT116 p53-/- cells. ISYNA1 mRNA expression in primary RCC were significantly correlated to OS and PFS ( p < 0.01 ). CONCLUSIONS We identified ISYNA1 as a novel p53 target gene, and unveiled to regulate intracellular myo-inositol content by p53-ISYNA1 pathway. Our findings shew that ISYNA1 mRNA expression in primary renal cancer was correlated to overall survival and progression free survival. Therefore, we considered that ISYNA1 might be useful as a novel prognostic factor for the post-operative metastasis. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1200 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Tomoyuki Koguchi More articles by this author Kei Ishibashi More articles by this author Akifumi Onagi More articles by this author Ryo Tanji More articles by this author Ruriko Takinami More articles by this author Seiji Hoshi More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Hidenori Akaihata More articles by this author Masao Kataoka More articles by this author Soichiro Ogawa More articles by this author Nobuhiro Haga More articles by this author Ken Aikawa More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP39-13 IDENTIFICATION OF ISYNA1 AS A POTENTIAL PROGNOSTIC BIOMARKER FOR RENAL CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-13 IDENTIFICATION OF ISYNA1 AS A POTENTIAL PROGNOSTIC BIOMARKER FOR RENAL CANCER Tomoyuki Koguchi, Kanako Matsuoka, Junya Hata, Yuichi Sato, Hidenori Akaihata, Masao Kataoka, Soichiro Ogawa, Nobuhiro Haga, Nobuhiro Kushida, Kei Ishibashi, Ken Ikawa, and Yoshiyuki Kojima Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Kanako MatsuokaKanako Matsuoka More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Hidenori AkaihataHidenori Akaihata More articles by this author , Masao KataokaMasao Kataoka More articles by this author , Soichiro OgawaSoichiro Ogawa More articles by this author , Nobuhiro HagaNobuhiro Haga More articles by this author , Nobuhiro KushidaNobuhiro Kushida More articles by this author , Kei IshibashiKei Ishibashi More articles by this author , Ken IkawaKen Ikawa More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1187AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In response to cellular stress, p53 exerts tumor-suppressive effects, such as apoptosis, cell cycle arrest, and senescence, through the induction of its target genes. Recently, p53 has been shown to control cellular homeostasis by regulating energy metabolism, glycolysis, antioxidant effects, and autophagy. A previous study has reported an association between the p53 expression pattern in immunohistochemical analyses of tumor and the prognosis of renal cell carcinoma (RCC). Although various tumors have p53 mutations, RCC has few p53 mutations. As such, we examined whether a p53 target gene might affect the prognosis of RCC patients. We identified a novel p53 target and examined whether it was useful as a prognostic factor for renal cancer. METHODS To screen for novel p53 target genes, we conducted a cDNA microarray analysis using mRNAs isolated from HCT116 p53+/+ and HCT116 p53-/- cells that were treated with 2 μg/ml of Adriamycin. To investigate whether mRNA transcription is regulated by p53, we performed a reporter assay and a chromatin immunoprecipitation (ChIP) assay using U373MG cells. To evaluate the effect of the novel p53 target on the biosynthesis of myo-inositol (MI), we performed a MI assay using HEK293T cells that were transfected with a plasmid expressing the novel p53 target, or HCT116 p53+/+ and HCT116 p53-/- cells that were treated with Adriamycin. To examine whether the novel p53 target would be useful as a prognosis factor for renal cancer, we performed a prognostic analysis using the mRNA expression ratio (tumor / normal kidney tissues) of the novel p53 target in RCC. RESULTS The results of the cDNA microarray analysis revealed inositol 3-phosphate synthase 1 ( ISYNA1 ) as a novel candidate gene. From the results of the reporter assay and the ChIP assay, we found p53 response elements in the promoter region and the seventh exon. As such, ISYNA1 was identified as a novel p53 target. The results of MI assay showed that ISYNA1 expression increased myo-inositol levels in the cells. In addition, DNA damage significantly increased the intracellular MI content in HCT116 p53+/+ cells, but did not affect the MI content in HCT116 p53-/- cells. The ISYNA1 mRNA expression ratios in renal cancer and renal clear cell carcinoma were significantly correlated with overall survival (p < 0.01). CONCLUSIONS We identified ISYNA1 as a novel p53 target gene and found that it regulated the intracellular MI content via a p53-ISYNA1 pathway. Our findings show that the ISYNA1 mRNA expression ratio is correlated to overall survival in RCC. Therefore, ISYNA1 might be useful as a novel prognostic factor for renal cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e498 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Tomoyuki Koguchi More articles by this author Kanako Matsuoka More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Hidenori Akaihata More articles by this author Masao Kataoka More articles by this author Soichiro Ogawa More articles by this author Nobuhiro Haga More articles by this author Nobuhiro Kushida More articles by this author Kei Ishibashi More articles by this author Ken Ikawa More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Involvement of Inositol Biosynthesis and Nitric Oxide in the Mediation of UV-B Induced Oxidative Stress

The involvement of NO-signaling in ultraviolet B (UV-B) induced oxidative stress (OS) in plants is an open question. Inositol biosynthesis contributes to numerous cellular functions, including the regulation of plants tolerance to stress. This work reveals the involvement of inositol-3-phosphate synthase 1 (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives, in the response to NO-dependent OS in Arabidopsis. Homozygous mutants deficient for IPS1 (atips1) and wild-type plants were transformed with a reduction- grx1-rogfp2 and used for the dynamic measurement of UV-B-induced and SNP (sodium nitroprusside)-mediated oxidative stresses by confocal microscopy. atips1 mutants displayed greater tissue-specific resistance to the action of UV-B than the wild type. SNP can act both as an oxidant or repairer depending on the applied concentration, but mutant plants were more tolerant than the wild type to nitrosative effects of high concentration of SNP. Additionally, pretreatment with low concentrations of SNP (10, 100 μM) before UV-B irradiation resulted in a tissue-specific protective effect that was enhanced in atips1. We conclude that the interplay between nitric oxide and inositol signaling can be involved in the mediation of UV-B-initiated oxidative stress in the plant cell.

Overexpression of IbMIPS1 gene enhances salt tolerance in transgenic sweetpotato

AbstractMyo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in the de novo biosynthesis of myo-inositol in plants. In the present study, the IbMIPS1 gene was introduced into sweetpotato cultivar Xushu 18 and the transgenic plants exhibited significantly enhanced salt tolerance compared with the wild-type (WT). Overexpression of IbMIPS1 up-regulated the salt stress responsive genes, including myo-inositol monophosphatase (MIPP), pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), psbA, phosphoribulokinase (PRK), and superoxide dismutase (SOD) genes, under salt stress. Inositol and proline content, SOD and photosynthesis activities were significantly increased, whereas malonaldehyde (MDA) and H2O2 contents were significantly decreased in the transgenic plants. These findings suggest that the IbMIPS1 gene may enhance salt tolerance of sweetpotato by regulating the expression of salt stress responsive genes, increasing the content of inositol and proline and enhancing the activity of photosynthesis.

Plasmodium falciparum is dependent on de novo myo‐inositol biosynthesis for assembly of GPI glycolipids and infectivity

Intra-erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo-inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo-inositol-3-phosphate, indicating increased de novo biosynthesis of myo-inositol from glucose 6-phosphate. Metabolic labelling studies with (13) C-U-glucose in the presence and absence of exogenous inositol confirmed that de novo myo-inositol synthesis occurs in parallel with myo-inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo-inositol was used to synthesize bulk PI, only de novo-synthesized myo-inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo-inositol 3-phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo-inositol biosynthesis for assembly of a sub-pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.

Biosynthesis of myo-inositol in lycopods: characteristics of the pteridophytic l-myo-inositol-1-phosphate synthase and myo-inositol-1-phosphate phosphatase from the strobili of Lycopodium clavatum and Selaginella monospora

Two key enzymes of myo-inositol metabolism [l-myo-inositol-1-phosphate synthase (MIPS) and myo-inositol-1-phosphate-phosphatase (MIPP)] were identified for the first time in Pteridophyta in Lycopodium clavatum and Selaginella monospora. Both enzymes from both plants were partly purified and the degree of purity obtained from 47 to 75 folds. MIPS preparations specifically utilized d-glucose-6-phosphate and NAD+ as its substrate and coenzyme with the K m values for G-6-P and NAD+ were 1.74 and 0.34 mM in L. clavatum; 2.32 and 0.37 mM in S. monospora. MIPP preparations used d/l-myo-inositol-1-phosphate as its principal substrate with the K m values for MIP was 0.068 mM in L. clavatum and 0.076 mM in S. monospora. The pH reliance of all the preparations was around 7.0–7.5 and different cations had variable role.

Down-regulation of UDP-glucuronic Acid Biosynthesis Leads to Swollen Plant Cell Walls and Severe Developmental Defects Associated with Changes in Pectic Polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

The synergistic effects of sugar and abscisic acid on myo-inositol-1-phosphate synthase expression.

1L-myo-inositol-1-phosphate [Ins(1)P1] synthase (EC 5.5.1.4) catalyses the formation of Ins(1)P1 from glucose-6-phosphate, the first step in the biosynthesis of myo-inositol. Ins(1)P1 is a precursor of phytin (inositol hexakisphosphate), a storage form of phosphate and cations in seeds. Since sucrose and abscisic acid (ABA) are known to affect synthesis of storage compounds in seeds, we investigated the effects of ABA and sucrose on Ins(1)P1 synthase gene (RINO1) expression in cultured cells derived from the scutellum of mature rice seeds. Higher levels of RINO1 transcript accumulation were evident after treatment with either sucrose (10-100 mM) or ABA (10-8 M to 10-4 M). Glucose was also effective in the upregulation, whereas mannitol was not, suggesting that sucrose and glucose acted as metabolizable sugars and not as osmotica. Treatment with ABA and sucrose together resulted in much higher levels of transcript accumulation, suggesting a synergistic induction of the Ins(1)P1 synthase gene.

Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis.

Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.

Reduced absolute rate of myo-inositol biosynthesis of cultured bovine retinal capillary pericytes in high glucose

De novo biosynthesis of myo-inositol (MI) by permeabilized cultured bovine retinal capillary pericytes (BRCP) and feline retinal pigment epithelial cells (FRPE), grown in different concentrations of glucose, were studied. After incubation with a physiological concentration of [ 14C]glucose 6-phosphate (G6P), the radioactive G6P derivatives were quantitated by a single HPLC column. Based on the determined specific activity of [ 14C]G6P, activities of inositol 1-phosphate synthase (MI synthase) were calculated. The activity of MI synthase was reduced 48% by growing BRCP in a high-glucose medium (20 m m) in comparison with that in the normal medium (glucose 5m m). In contrast, the de novo MI biosynthesis by FRPE was not changed with increasing concentrations of glucose in the medium. As compared with MI uptake previously studied, the synthesized MI contributes a substantial proportion of cellular MI pool in BRCP. Therefore, in BRCP growing in high glucose the reduced MI biosynthesis aggravates the low MI content resulting from the inhibited MI uptake, and thus leads to altered inositol phospholipid metabolism.

Measurement of biosynthesis of myo-inositol from glucose 6-phosphate

Publisher Summary This chapter provides an overview of the measurement of the biosynthesis of myo-inositol from glucose 6-phosphate. Myo-inositol, the principal cyclohexanehexol in nature and the precursor of the inositol phospholipids, is synthesized in two stages from glucose 6-phosphate. As in the other fates of glucose 6-phosphate leading to various classes of sugars, the first stage is an isomerization; the product is L-myo-inositol 1-phosphate. The reaction is catalyzed by a specific enzyme, L-myo-inositol-l-phosphate synthase with an absolute requirement for NAD + . The second stage is catalyzed by a phosphatase requiring Mg 2+ . Although the enzyme is distinct from the large classes of nonspecific phosphatases, it cannot differentiate between the enantiomeric forms of myoinositol 1-phosphate. The chapter presents various measurement methods. Myo-inositol 1-phosphate can be measured in several ways, the choice depending on the situation confronting the investigator. The various methods are chemical, enzymatic, and chromatographic.

Tissue Content and Metabolism of myo-Inositol in Normal and Lipodystrophic Gerbils

Experiments were conducted to evaluate the effect of diet and sex difference on the development of an intestinal lipodystrophy due to myo-inositol deficiency. Tissue contents of free and lipid-bound myo-inositol as well as the activities of L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) and phosphatase (EC 3.1.3.25), and myo-inositol oxygenase (EC 1.13.99.1) were determined in male and female gerbils under various conditions. The enzyme study proved that the essentiality of dietary myoinositol for this species was not due to the lack of such enzyme activity. The lower susceptibility of male gerbils to myo-inositol deficiency could be explained by the contribution of the biosynthesis of myo-inositol in the testis, as shown by a difference between intact and castrated animals. Although feeding coconut oil to the myo-inositol-deficient female gerbils produced greater myo-inositol depletion as well as a more severe intestinal lesion than the feeding of safflower oil, the difference in myo-inositol status could be only in part responsible for different degrees of lipodystrophy. Additionally, neither dietary type of fat nor exogenous myo-inositol altered the activities of either hepatic or intestinal synthase and phosphatase, or kidney oxygenase. Thus, this study indicates that both sex and dietary factors might influence myo-inositol status to varying extents, but the diet-induced change in tissue myo-inositol was not reflected by the enzyme activity as measured in vitro.