The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.     

Spin labelled phosphatidyl-MYO-inositols: Synthesis and properties

The paper describes the synthesis of phosphatidyl-myo-inositol mono and bi-radicals, containing spin labelled stearic acid residues, which can be used in studies of membranes and protein lipid interactions. The EPR study of certain physico-chemical properties of the products important for their application as spin probes (aggregation in various media, pH stability range) is reported.     

Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the “open” and “closed” conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal “Rossmann fold” domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.     

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na⁺- or H⁺-linked myo-inositol transporters. While Na⁺-coupled myo-inositol transporters are found exclusively in the plasma membrane, H⁺-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H⁺-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.     

Ppm1-Encoded Polyprenyl Monophosphomannose Synthase Activity Is Essential for Lipoglycan Synthesis and Survival in Mycobacteria

The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria: IMPLICATIONS FOR CATALYSIS*

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the β-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic α-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response, host-pathogen interactions, and tumor cell metastasis. Most of the enzymes encoded in eukaryotic/prokaryotic/archaeans genomes that are responsible for the biosynthesis and modification of glycan structures are GTs³ (1). Here we have focused in the phosphatidyl-myo-inositol mannosyltransferase A (PimA), an essential enzyme of mycobacterial growth that initiates the biosynthetic pathway of key structural elements and virulence factors of Mycobacterium tuberculosis, the phosphatidyl-myo-inositol mannosides (PIM) lipomannan and lipoarabinomannan (2–5). This amphitropic enzyme catalyzes the transfer of a Manp residue from GDP-Man to the 2-position of PI to form phosphatidyl-myo-inositol monomannoside (PIM₁) on the cytoplasmic side of the plasma membrane (2) (Fig. 1).Open in a separate windowFIGURE 1.PIM₁ biosynthesis in mycobacteria. PimA transfers a Manp residue from GDP-Man to the 2-position of the myo-inositol ring of PI to form PIM₁ (where DAG is di-acyl-glycerol, and INS-P is 1-l-myo-inositol phosphate). The reaction occurs with retention of the anomeric configuration of the sugar donor.Although considerable progress has been made in recent years in understanding the mode of action of GTs at the molecular level, the mechanisms that govern recognition of lipid acceptors and membrane association of peripheral membrane-binding GTs remains poorly understood. GTs can be classified as either “inverting” or “retaining” enzymes according to the anomeric configuration of the reaction substrates and products. A single displacement mechanism in which a general base assists in the activation of the acceptor substrate for nucleophilic attack by the sugar donor is well established for inverting enzymes (6, 7). In contrast, the catalytic mechanism for retaining enzymes, including PimA, remains unclear. By analogy with glycosylhydrolases, a double displacement mechanism via the formation of a covalent glycosyl-enzyme intermediate was first proposed (8). However, in the absence of direct evidence of a viable covalent intermediate, an alternative mechanism known as the S_Ni “internal return” has been suggested where phospho-sugar donor bond breakage and sugar-acceptor bond formation occur in a concerted, but necessarily stepwise manner on the same face of the sugar (6, 9). Only two protein topologies have been found for nucleotide-diphospho-sugar-dependent enzymes among the first 30 GT sequence-based families (10) (see the carbohydrate-active enzymes (CAZy) data base) for which three-dimensional structures have been reported (11). These topologies are variations of “Rossmann-like” domains and have been defined as GT-A (12) and GT-B (13). Both inverting and retaining enzymes were found in GT-A and GT-B folds, indicating that there is no correlation between the overall fold of GTs and their catalytic mechanism. The primary sequence of PimA contains the GPGTF (glycogen phosphorylase/GT) motif, a signature present in enzymes of the GT-B fold (14). GT-B proteins do not use divalent cations and consist of two Rossmann-like (β-α-β) domains separated by a deep fissure. Therefore, an important interdomain movement has been predicted in some members of this superfamily during catalysis, including MurG (15), glycogen synthase (16),and the myo-inositol 1-phosphate N-acetylglucosaminyltransferase MshA (17).To perform their biochemical functions, membrane-binding GTs interact with membranes by two different mechanisms. Whereas integral membrane GTs are permanently attached through transmembrane regions (e.g. hydrophobic α-helices) (18) peripheral membrane-binding GTs temporarily bind membranes by (i) a stretch of hydrophobic residues exposed to bulk solvent, (ii) electropositive surface patches that interact with acidic phospholipids (e.g. amphipathic α-helices), and/or (iii) protein-protein interactions (19–22). A close interaction of the enzyme with membranes might be a strict requirement for PI modification by PimA. We recently solved the crystal structure of PimA from Mycobacterium smegmatis (MsPimA) in complex with the donor substrate GDP-Man (23, 24). The notion of a membrane-associated protein via electrostatic interactions is consistent with the finding of an amphipathic α-helix and surface-exposed hydrophobic residues in the N-terminal domain of MsPimA. Despite the fact that sugar transfer is catalyzed between the mannosyl group of the GDP-Man donor and the myo-inositol ring of PI, the enzyme displays an absolute requirement for both fatty acid chains of PI in order for the transfer reaction to take place. Furthermore, PimA was able to bind monodisperse PI, but its transferase activity was stimulated by high concentrations of nonsubstrate anionic surfactants, indicating that the reaction requires a lipid-water interface. We thus proposed a model of interfacial catalysis in which PimA recognizes the fully acylated substrate PI with its polar head within the catalytic cleft and the fatty acid moieties only partially sequestered from the bulk solvent (24).This study describes a detailed investigation of the lipid acceptor binding site and the conformational properties of PimA in solution. Using a combination of limited proteolysis, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), analytical ultracentrifugation (AUC) and site-directed mutagenesis, we propose a plausible model for substrates recognition and binding. The implications of this model for the comprehension of the early steps of PIM biosynthesis and the catalytic mechanism of other members of the peripheral membrane-binding GT family are discussed.     

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria: PimB�� IS AN ESSENTIAL ENZYME OF MYCOBACTERIUM SMEGMATIS*

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB′ (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB′ exclusively transfers to the 6-position. Moreover, whereas PimB′ can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM₁) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM₁ product of PimB′. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB′ indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB′. Importantly, genetic evidence is provided that pimB′ is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac₁PIM₂, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB′, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.PIMs³ are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes.⁴ They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3–6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc²155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimB′, the enzyme responsible for the transfer of the second Manp to the 6-position of the myo-Ins ring of PIM₁, still remains controversial. The Rv0557 protein of Mycobacterium tuberculosis H37Rv (PimB; MSMEG_1113 in M. smegmatis mc²155) was originally characterized as PimB′ (7). However, the lack of an Rv0557 ortholog in the genome of Mycobacterium leprae and the fact that the disruption of this gene in M. tuberculosis Erdman did not significantly affect the biosynthesis of PIMs suggest that compensatory activities exist in the bacterium or that Rv0557 serves another primary function (8, 9). Somewhat supporting the latter hypothesis, the ortholog of Rv0557 in Corynebacterium glutamicum (NCgl0452, renamed mgtA) was implicated in the mannosylation of a novel glycolipid (1,2-di-O-C₁₆/C_18:1-(α-d-mannosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol), and Rv0557 from M. tuberculosis was reported to functionally complement for this enzyme in a C. glutamicum knock-out mutant (10). However, to our knowledge this mannosylated glycolipid has never been reported in mycobacteria, and it remains unclear whether PimB serves a similar physiological function in Mycobacterium spp.More recently, Lea-Smith et al. (11) have shown that the biosynthesis of Ac₁PIM₂ from Ac₁PIM₁ in C. glutamicum is catalyzed by NCgl2106 (Cg-PimB′). Disruption of the NCgl2106 gene totally abolished Ac₁PIM₂ production in the mutant, arguing against the existence of a compensatory activity associated with the corynebacterial PimB enzyme. Although Ac₁PIM₂ production in Cg-pimB′ and Cg-pimB′/Cg-pimB knock-out mutants was restored upon complementation with the M. tuberculosis Rv2188c gene (11, 12), direct evidence that Rv2188c carried out the same physiological function in mycobacteria has been lacking. Moreover, in light of the recent work by Torrelles et al. (9) showing an involvement of pimB (Rv0557) in the synthesis of LM and LAM in M. tuberculosis Erdman and of the demonstrated relaxed substrate specificity of the M. tuberculosis PimB (Rv0557) and PimB′ (Rv2188c) enzymes expressed in C. glutamicum (12), whether or not pimB and pimB′ could compensate for one another in mycobacteria remained open to speculation.Both PIM₁ and PIM₂ can be acylated with palmitate at position 6 of the Manp residue transferred by PimA by the acyltransferase MSMEG_2934 (orthologous to Rv2611c from M. tb) to form Ac₁PIM₁ and Ac₁PIM₂, respectively (13). Ac₁PIM₂ can further be acylated at position 3 of the myo-Ins ring by an as yet unidentified acyltransferase to yield Ac₂PIM₂. Importantly, Ac₁PIM₂ and Ac₂PIM₂ are among the most abundant forms of PIMs found in mycobacteria and are considered both metabolic end products and intermediates in the biosynthesis of more polar forms of PIMs (PIM₅ and PIM₆), LM, and LAM.In this work, clear evidence is provided that PimB′ (MSMEG_4253 in M. smegmatis mc²155) is the α-ManT responsible for the biosynthesis of PIM₂ from PIM₁ in mycobacteria and that no other ManT can compensate for a deficiency in this enzyme in M. smegmatis. Like PimA (5), PimB′ is essential to the growth of M. smegmatis. Cell-free assays using purified PimA and PimB′ and M. smegmatis membrane preparations provide new insights into the sequential events leading to the synthesis of the early forms of PIMs in mycobacteria.     

Occurrence of 1-Glyceryl-1-myo-Inosityl Phosphate in Hyperthermophiles

The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of α- and β-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while α- and β-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.     

Metabolic engineering of Corynebacterium glutamicum for production of scyllo-inositol,a drug candidate against Alzheimer's disease

Scyllo-inositol has been identified as a potential drug for the treatment of Alzheimer's disease. Therefore, cost-efficient processes for the production of this compound are desirable. In this study, we analyzed and engineered Corynebacterium glutamicum with the aim to develop competitive scyllo-inositol producer strains. Initial studies revealed that C. glutamicum naturally produces scyllo-inositol when cultured with myo-inositol as carbon source. The conversion involves NAD⁺-dependent oxidation of myo-inositol to 2-keto-myo-inositol followed by NADPH-dependent reduction to scyllo-inositol. Use of myo-inositol for biomass formation was prevented by deletion of a cluster of 16 genes involved in myo-inositol catabolism (strain MB001(DE3)Δiol1). Deletion of a second cluster of four genes (oxiC-cg3390-oxiD-oxiE) related to inositol metabolism prevented conversion of 2-keto-myo-inositol to undesired products causing brown coloration (strain MB001(DE3)Δiol1Δiol2). The two chassis strains were used for plasmid-based overproduction of myo-inositol dehydrogenase (IolG) and scyllo-inositol dehydrogenase (IolW). In BHI medium containing glucose and myo-inositol, a complete conversion of the consumed myo-inositol into scyllo-inositol was achieved with the Δiol1Δiol2 strain. To enable scyllo-inositol production from cheap carbon sources, myo-inositol 1-phosphate synthase (Ino1) and myo-inositol 1-phosphatase (ImpA), which convert glucose 6-phosphate into myo-inositol, were overproduced in addition to IolG and IolW using plasmid pSI. Strain MB001(DE3)Δiol1Δiol2 (pSI) produced 1.8 g/L scyllo-inositol from 20 g/L glucose and even 4.4 g/L scyllo-inositol from 20 g/L sucrose within 72 h. Our results demonstrate that C. glutamicum is an attractive host for the biotechnological production of scyllo-inositol and potentially further myo-inositol-derived products.     

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.     

Unimpaired formation of hormone-stimulated inositol triphosphate in human mesangial cells under hyperglycemic conditions

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grwon under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (K_m values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-triphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol triphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Defective Craniofacial Development and Brain Function in a Mouse Model for Depletion of Intracellular Inositol Synthesis

myo-Inositol is an essential biomolecule that is synthesized by myo-inositol monophosphatase (IMPase) from inositol monophosphate species. The enzymatic activity of IMPase is inhibited by lithium, a drug used for the treatment of mood swings seen in bipolar disorder. Therefore, myo-inositol is thought to have an important role in the mechanism of bipolar disorder, although the details remain elusive. We screened an ethyl nitrosourea mutant mouse library for IMPase gene (Impa) mutations and identified an Impa1 T95K missense mutation. The mutant protein possessed undetectable enzymatic activity. Homozygotes died perinatally, and E18.5 embryos exhibited striking developmental defects, including hypoplasia of the mandible and asymmetric fusion of ribs to the sternum. Perinatal lethality and morphological defects in homozygotes were rescued by dietary myo-inositol. Rescued homozygotes raised on normal drinking water after weaning exhibited a hyper-locomotive trait and prolonged circadian periods, as reported in rodents treated with lithium. Our mice should be advantageous, compared with those generated by the conventional gene knock-out strategy, because they carry minimal genomic damage, e.g. a point mutation. In conclusion, our results reveal critical roles for intracellular myo-inositol synthesis in craniofacial development and the maintenance of proper brain function. Furthermore, this mouse model for cellular inositol depletion could be beneficial for understanding the molecular mechanisms underlying the clinical effect of lithium and myo-inositol-mediated skeletal development.     

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 dehydrogenase and scyllo-inositol dehydrogenase from Lactobacillus casei BL23 bind their substrates in very different orientations

Many bacteria can use myo-inositol as the sole carbon source using enzymes encoded in the iol operon. The first step is catalyzed by the well-characterized myo-inositol dehydrogenase (mIDH), which oxidizes the axial hydroxyl group of the substrate to form scyllo-inosose. Some bacteria, including Lactobacillus casei, contain more than one apparent mIDH-encoding gene in the iol operon, but such redundant enzymes have not been investigated. scyllo-Inositol, a stereoisomer of myo-inositol, is not a substrate for mIDH, but scyllo-inositol dehydrogenase (sIDH) enzymes have been reported, though never observed to be encoded within the iol operon. Sequences indicate these enzymes are related, but the structural basis by which they distinguish their substrates has not been determined. Here we report the substrate selectivity, kinetics, and high-resolution crystal structures of the proteins encoded by iolG1 and iolG2 from L. casei BL23, which we show encode a mIDH and sIDH, respectively. Comparison of the ternary complex of each enzyme with its preferred substrate reveals the key variations allowing for oxidation of an equatorial versus an axial hydroxyl group. Despite the overall similarity of the active site residues, scyllo-inositol is bound in an inverted, tilted orientation by sIDH relative to the orientation of myo-inositol by mIDH.     

myo-Inositol transport in plasma membrane of rat kidney

The myo-inositol transport system in kidney plasma mambrane preparation was investigated. myo-Inisitol uptake was more rapid than that due to non-specific uptake. Specific myo-inisitol uptake was temperature dependent and pH sensitive; the optimum was at pH 7.4. Specific myo-insitol uptake was inhibited by scyllitol and inosose-2 but not(+)-inositol, d-glucose, d-galactose or mannitol. Inhibition of myo-inositol uptake by scyllitol was of the competitive type. It showed that the transport system is stereospecific and that myo-inositol shares the transport system with scyllitol. Moreover, the specific myo-inositol uptake was inhibited by phlorizin. Counter transport of myo-inositol was demonstrated. The results indicate that myo-inositol uptake by the membrane preparation represents the entry into the intravesicular spaces rather than binding to the membrane.It was concluded that the plasma membrane of rat kidney has a cyclitol carrier system specific to myo-inositol and scyllitol.     

myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination

Radiolabeled d-[1-³H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the ³H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained ³H, showing that de novo synthesis of myo-inositol from [1-³H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The ³H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, ³H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.     

A sensitive radioisotopic assay of myo-inositol: Its application to rat pancreatic islets

A radioisotopic procedure for the assay of myo-inositol is presented. It is based on the generation of NADH from NAD⁺ in the reaction catalyzed by myo-inositol dehydrogenase and the subsequent NADH-dependent conversion of 2-[U-¹⁴C]ketoglutarate to ¹⁴C-labeled l-glutamate in the reaction catalyzed by glutamate dehydrogenase. This method was applied to the measurement of myo-inositol in rat pancreatic islets. The myo-inositol islet content was decreased when the animals were fed a diet deprived of myo-inositol. When incubated in the absence of exogenous d-glucose, pancreatic islets, like parotid cells, released myo-inositol in the incubation medium. Over 90 min of incubation, a rise in extracellular d-glucose concentration increased the myo-inositol islet content, which was decreased, however, after incubation in the presence of carbamylcholine. These findings indicate that the myo-inositol content of islets is affected by nutritional and other environmental factors.