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1.  PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling 
PLoS ONE  2009;4(3):e4911.
Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor -κB (NF-κB) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-κB to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.
PMCID: PMC2654112  PMID: 19290049
2.  Acceptor Substrate Discrimination in Phosphatidyl-myo-inositol Mannoside Synthesis 
The Journal of Biological Chemistry  2010;285(48):37741-37752.
Long term survival of the pathogen Mycobacterium tuberculosis in humans is linked to the immunomodulatory potential of its complex cell wall glycolipids, which include the phosphatidylinositol mannoside (PIM) series as well as the related lipomannan and lipoarabinomannan glycoconjugates. PIM biosynthesis is initiated by a set of cytosolic α-mannosyltransferases, catalyzing glycosyl transfer from the activated saccharide donor GDP-α-d-mannopyranose to the acceptor phosphatidyl-myo-inositol (PI) in an ordered and regio-specific fashion. Herein, we report the crystal structure of mannosyltransferase Corynebacterium glutamicum PimB′ in complex with nucleotide to a resolution of 2.0 Å. PimB′ attaches mannosyl selectively to the 6-OH of the inositol moiety of PI. Two crystal forms and GDP- versus GDP-α-d-mannopyranose-bound complexes reveal flexibility of the nucleotide conformation as well as of the structural framework of the active site. Structural comparison, docking of the saccharide acceptor, and site-directed mutagenesis pin regio-selectivity to a conserved Asp residue in the N-terminal domain that forces presentation of the correct inositol hydroxyl to the saccharide donor.
PMCID: PMC2988379  PMID: 20843801
Cell Wall; Glycoconjugate; Membrane Lipids; Protein Structure; X-ray Crystallography; Corynebacterium glutamicum; Mycobacterium tuberculosis; Glycosyltransferase
3.  Crystallization and preliminary crystallographic analysis of PimA, an essential mannosyltransferase from Mycobacterium smegmatis  
Mycobacterial PimA is an essential enzyme that catalyses the first mannosylation step in phosphatidyl-myo-inositol mannoside (PIM) biosynthesis. Crystals of the enzyme from M. smegmatis, obtained in the presence of GDP and myo-inositol, are orthorhombic (P212121) and diffract X-rays to 2.4 Å resolution.
Phosphatidylinositol mannosyltransferase (PimA) is an essential enzyme for mycobacterial growth that catalyses the first mannosylation step in phosphatidyl-myo-inositol mannoside (PIM) biosynthesis. The enzyme belongs to the large GT4 family of glycosyltransferases, for which no structure is currently available. Recombinant purified PimA from Mycobacterium smegmatis has been crystallized in the presence of GDP and myo-inositol. The crystals belong to space group P212121, with unit-cell parameters a = 37.2, b = 72.4, c = 138.2 Å, and diffract to 2.4 Å resolution.
PMCID: PMC1952298  PMID: 16511084
GT4 glycosyltransferase; tuberculosis; phosphatidylinositol mannoside; PIM; mycobacteria
4.  Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms 
PLoS ONE  2011;6(9):e24631.
Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM1 isomer and PIM2 mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM1 and PIM2 analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM1 and PIM2 analogues. CD14 was dispensable for PIM1 and PIM2 analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM1 and PIM2 analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.
PMCID: PMC3174970  PMID: 21949737
5.  Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant 
Antonie Van Leeuwenhoek  2008;94(2):277-287.
The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac1PIM2) resulting in the accumulation of Ac1PIM1 and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed ‘Cg-LM-A’) and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed ‘Cg-LM-B’. Further chemical analyses established the lipoglycan possessed an α-d-glucopyranosyluronic acid-(1 → 3)-glycerol (GlcAGroAc2) based anchor which was then further glycosylated by 8–22 mannose residues, with Man12–20GlcAGroAC2 molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-α by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity.
PMCID: PMC2480597  PMID: 18421567
Corynebacterium glutamicum; Lipomannan; Mannosyltransferase; PimB′
6.  Identification of a novel α(1→6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant 
Molecular Microbiology  2008;68(6):1595-1613.
Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C16/C18:1-(α-D-glucopyranosyluronic acid)-(1→3)-glycerol (GlcAGroAc2) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac1PIM2) and Man1GlcAGroAc2 were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[14C]Man established that C. glutamicum synthesized a novel α(1→6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac1PIM2 and Man1GlcAGroAc2 respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core α(1→6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac1PI[14C]M2 and [14C]Man1GlcAGroAc2 primers respectively. Use of the acceptor α-D-Manp-(1→6)-α-D-Manp-O-C8 in an in vitro cell-free assay confirmed NCgl1505 as an α(1→6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroα(1→6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.
PMCID: PMC2440535  PMID: 18452585
7.  Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death 
Glycobiology  2009;19(7):743-755.
The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal α-[1→2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.
PMCID: PMC2688391  PMID: 19318518
lipoarabinomannan; macrophage death; mannosyltransferase; Mycobacterium tuberculosis; phosphatidyl-myo-inositol mannoside
8.  Structural Characterization of Phosphatidyl-myo-Inositol Mannosides from Mycobacterium bovis Bacillus Calmette Gúerin by Multiple-Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization. II. Monoacyl- and Diacyl-PIMs 
The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M – H – diacylglycerol]−) in the MS2-spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e., R3CO2H) from that on the inositol (i.e., R4CO2H) is based on the findings that the MS3-spectrum of [M – H – diacylglycerol]− contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e., the [M – H – diacylglycerol – R3CO2H]− ion), while the ion arising from loss of the fatty acid substituent at the inositol (i.e., the [M – H – diacylglycerol – R4CO2H]− ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS4 of the [M – H – diacylglycerol – R3CO2H]− ion, which gives rise to a prominent ion corresponding to loss of R4CO2H. An [M – H – acylmannose]− ion was also observed in the MS2-spectra and, thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS2, MS3, and MS4 permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guérin.
PMCID: PMC2044505  PMID: 17141525
9.  Activation of IP3 receptors by synthetic bisphosphate ligands† †Dedicated to Dr Melanie N. Trusselle (1973–2008).  
Combining synthesis and mutagenesis, we show that a cation–π interaction between adenine of adenophostin analogues and Arg504 of IP3 receptors (IP3R) is responsible for the enhanced activity of adenophostins and can replace a phosphate–receptor interaction.
Ca2+ release by d-myo-inositol 1,4,5-trisphosphate receptors (IP3Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP3, with P-5 and P-4 engaging the α and β domains of the binding site; using synthesis and mutagenesis we show that the adenine of synthetic glyconucleotides, through an interaction with Arg504, can replace the interaction of either P-1 or P-5 with the α-domain producing, respectively, the most potent bisphosphate agonist yet synthesised and the first agonist of IP3R without a vicinal bisphosphate motif; this will stimulate new approaches to IP3R ligand design.
PMCID: PMC2898634  PMID: 19240874
10.  Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate 
The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of the changes in cell physiology mediated by these lipids, three new metabolically-stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-monofluoromethylphosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl and dibutyryl chains. In addition, we introduce a new phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of “pKa-matched” monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labelled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis.
PMCID: PMC2535791  PMID: 16417379
11.  Biosynthetic Pathways of Inositol and Glycerol Phosphodiesters Used by the Hyperthermophile Archaeoglobus fulgidus in Stress Adaptation▿ †  
Journal of Bacteriology  2006;188(23):8128-8135.
Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into l-myo-inositol 1-phosphate by l-myo-inositol 1-phosphate synthase; (ii) l-myo-inositol 1-phosphate was activated to CDP-inositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with l-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1′-di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and l-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X,1′X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.
PMCID: PMC1698214  PMID: 17028285
12.  Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis 
BMC Microbiology  2010;10:50.
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.
Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained.
We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.
PMCID: PMC2834668  PMID: 20167072
13.  Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages 
Annals of the Rheumatic Diseases  2001;60(Suppl 3):iii6-iii12.
BACKGROUND—Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-κB, AP1, and MAP kinases.
RESULTS—This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor α (TNFα), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFα, MIP-1β, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1β production.
CONCLUSION—Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-κB, AP1, MAP kinases, and PI-3-K.

PMCID: PMC1766663  PMID: 11890657
14.  A Functional myo-Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean (Glycine max [L.] Merr.) 
Journal of Bacteriology  2001;183(8):2595-2604.
Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.
PMCID: PMC95177  PMID: 11274120
15.  Evolution of the total syntheses of ustiloxin natural products and their analogues 
Ustiloxins A–F are antimitotic heterodetic cyclopeptides containing a 13–membered cyclic core structure with a synthetically challenging chiral tertiary alkyl–aryl ether linkage. The first total synthesis of ustiloxin D was achieved in 31 linear steps using an SNAr reaction. An nOe study of this synthetic product showed that ustiloxin D existed as a single atropisomer. Subsequently, highly concise and convergent syntheses of ustiloxins D and F were developed by utilizing a newly discovered ethynyl aziridine ring–opening reaction in a longest linear sequence of 15 steps. The approach was further optimized to achieve a better macrolactamization strategy. Ustiloxins D, F and eight analogues (14–MeO–ustiloxin D, four analogues with different amino acid residues at the C–6 position, and three (9R, 10S)–epi–ustiloxin analogues) were prepared via the second generation route. Evaluation of these compounds as inhibitors of tubulin polymerization demonstrated that variation at the C–6 position is tolerated to a certain extent. In contrast, the S configuration of the C–9 methylamino group and a free phenolic hydroxyl group are essential for inhibition of tubulin polymerization.
PMCID: PMC2935141  PMID: 18229928
16.  Ppm1-Encoded Polyprenyl Monophosphomannose Synthase Activity Is Essential for Lipoglycan Synthesis and Survival in Mycobacteria 
PLoS ONE  2012;7(10):e48211.
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.
PMCID: PMC3485146  PMID: 23118955
17.  Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+ 
The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear.
We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own.
The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.
PMCID: PMC2080633  PMID: 17725819
18.  The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol 
Molecular microbiology  2006;61(1):89-105.
In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-d-myo-inositol-3-phosphate, catalysed by a 1-d-myo-inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.
PMCID: PMC3793301  PMID: 16824097
19.  Occurrence and Role of Di-myo-Inositol-1,1′-Phosphate in Methanococcus igneus 
Applied and Environmental Microbiology  1994;60(10):3660-3664.
Methanococcus igneus, a hyperthermophilic marine methanogen (optimum growth temperature of 88°C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with l-α-glutamate and β-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-myo-inositol-1,1′-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (l-α-glutamate, β-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by 13CO2-pulse-CO2-chase experiments. The results indicated that both the β-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was l-α-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-myo-inositol-1,1′-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when M. igneus was grown at temperatures of ≥80°C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.
PMCID: PMC201870  PMID: 16349412
20.  Sodium/myo-Inositol Transporters: Substrate Transport Requirements and Regional Brain Expression in the TgCRND8 Mouse Model of Amyloid Pathology 
PLoS ONE  2011;6(8):e24032.
Inositol stereoisomers, myo- and scyllo-inositol, are known to enter the brain and are significantly elevated following oral administration. Elevations in brain inositol levels occur across a concentration gradient as a result of active transport from the periphery. There are two sodium/myo-inositol transporters (SMIT1, SMIT2) that may be responsible for regulating brain inositol levels. The goals of this study were to determine the effects of aging and Alzheimer's disease (AD)-like amyloid pathology on transporter expression, to compare regional expression and to analyze substrate requirements of the inositol transporters. QPCR was used to examine expression of the two transporters in the cortex, hippocampus and cerebellum of TgCRND8 mice, a mouse model of amyloid pathology, in comparison to non-transgenic littermates. In addition, we examined the structural features of inositol required for active transport, utilizing a cell-based competitive uptake assay. Disease pathology did not alter transporter expression in the cortex or hippocampus (p>0.005), with only minimal effects of aging observed in the cerebellum (SMIT1: F2,26 = 12.62; p = 0.0002; SMIT2: F2,26 = 8.71; p = 0.0015). Overall, brain SMIT1 levels were higher than SMIT2, however, regional differences were observed. For SMIT1, at 4 and 6 months cerebellar SMIT1 levels were significantly higher than cortical and hippocampal levels (p<0.05). For SMIT2, at all three ages both cortical and cerebellar SMIT2 levels were significantly higher than hippocampal levels (p<0.05) and at 4 and 6 months of age, cerebellar SMIT2 levels were also significantly higher than cortical levels (p<0.05). Inositol transporter levels are stably expressed as a function of age, and expression is unaltered with disease pathology in the TgCRND8 mouse. Given the fact that scyllo-inositol is currently in clinical trials for the treatment of AD, the stable expression of inositol transporters regardless of disease pathology is an important finding.
PMCID: PMC3162603  PMID: 21887366
21.  Rat L6 myotubes as an in vitro model system to study GLUT4-dependent glucose uptake stimulated by inositol derivatives 
Cytotechnology  2007;55(2-3):103-108.
Some of inositol derivatives have been reported to help the action of insulin stimulating glucose uptake in skeletal muscle cells. Rat L6 myotubes were employed in an attempt to develop an in vitro model system for investigation of the possible insulin-like effect of eight inositol derivatives, namely allo-inositol, d-chiro-inositol l-chiro-inositol, epi-inositol, muco-inositol, myo-inositol, scyllo-inositol and d-pinitol. At a higher concentration of 1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1 mM only d-chiro-inositol, l-chiro-inositol, epi-inositol and muco-inositol could induce glucose uptake, indicating their significant insulin-mimetic activity. Immunoblot analyses revealed that at least d-chiro-inositol, l-chiro-inositol, epi-inositol, muco-inositol and d-pinitol were able to induce translocation of glucose transporter 4 (GLUT4) to plasma membrane not only in L6 myotubes but also in skeletal muscles of rats ex vivo. These results demonstrated that L6 myotubes appeared efficient as an in vitro system to identify inositol derivatives exerting an insulin-like effect on muscle cells depending on the induced translocation of GLUT4.
PMCID: PMC2104555  PMID: 19002999
Glucose transporter 4; Glucose uptake; Inositol; L6 myotubes; Muscle cells
22.  Myo-inositol transport in Saccharomyces cerevisiae. 
Journal of Bacteriology  1982;150(2):441-446.
myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide.
PMCID: PMC216387  PMID: 7040334
23.  Quantitative analysis of myo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry 
Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC-MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.
PMCID: PMC3632838  PMID: 21856255
Inositol; glucose; mass spectrometry; urine; chromatography; epimers; HPLC-MS/MS
24.  Studies on the Biosynthesis of Streptomycin 
Applied Microbiology  1962;10(2):157-168.
Myo-inositol, especially in combination with arginine, enhances streptomycin production. Compounds which show structural relationship with myo-inositol are ineffective.
Myo-inositol decreases the incorporation of C14-glucose into streptomycin, particularly into streptidine. This effect suggests that myo-inositol is a precursor of the streptidine ring.
Methionine stimulates antibiotic production in a synthetic medium but proves to be unfavorable in a complex medium.
The γ- and δ-isomers of hexachlorocyclohexane inhibit streptomycin formation.
The formation of streptomycin by washed mycelium was studied. Essentially the same results were here obtained as with growing cultures.
PMCID: PMC1057830  PMID: 14468645
25.  Long-Chain Glycerol Diether and Polyol Dialkyl Glycerol Triether Lipids of Sulfolobus acidocaldarius 
Journal of Bacteriology  1974;119(1):106-116.
Cells of Sulfolobus acidocaldarius contain about 2.5% total lipid on a dry-weight basis. Total lipid was found to contain 10.5% neutral lipid, 67.6% glycolipid, and 21.7% polar lipid. The lipids contained C40H80 isopranol glycerol diethers. Almost no fatty acids were present. The glycolipids were composed of about equal amounts of the glycerol diether analogue of glucosyl galactosyl diglyceride and a glucosyl polyol glycerol diether. The latter compound contained an unidentified polyol attached by an ether bond to the glycerol diether. The polar lipids contained a small amount of sulfolipid, which appeared to be the monosulfate derivative of glucosyl polyol glycerol diether. About 40% of the lipid phosphorus was found in the diether analogue of phosphatidyl inositol. The remaining lipid phosphorus was accounted for by approximately equal amounts of two inositol monophosphate-containing phosphoglycolipids, inositolphosphoryl glucosyl galactosyl glycerol diether and inositolphosphoryl glucosyl polyol glycerol diether.
PMCID: PMC245579  PMID: 4407015

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