A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in methanoarchaeon Methanothermobacter thermautotrophicus cells.
ABSTRACT: Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[(14)C]inositol 1-phosphate, which was enzymatically prepared from [(14)C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.
Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.
Project description:Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in Bacteria and Archaea, and the basis for this domain-dependent specificity was investigated herein. The archaeon Archaeoglobus fulgidus and the bacterium Aquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and l-myo-inositol 1-phosphate. DIPPS of A. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of 31P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of A. fulgidus and Aq. aeolicus, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for sn-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for sn-glycerol 3-phosphate. In contrast, the partially purified A. fulgidus GCT uses sn-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e., A. fulgidus accumulates predominantly sn-glycero-1-phospho-3-l-myo-inositol, while Aq. aeolicus accumulates sn-glycero-3-phospho-3-l-myo-inositol. IMPORTANCE:Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and l-myo-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the Bacteria and Archaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require sn-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.
Project description:CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of D-serine with the enzyme was 30% of that observed for L-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in M. thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA. The substrate specificity of phosphatidylserine synthase from B. subtilis was quite similar to that observed for the M. thermautotrophicus archaetidylserine synthase, while the E. coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol. It was concluded that M. thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B. subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.
Project description:Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3'-phosphate-1'-phosphate from CDP-inositol and L-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K(m) values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V(max) determined at 90°C was 62.9 ?mol · min(-1) · mg of protein(-1). The structure of IPCT is composed of a central seven-stranded mixed ?-sheet, of which six ?-strands are parallel, surrounded by six ?-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X(4)-P-K. CTP, L-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.
Project description:The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg2+-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg2+ enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.
Project description:Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9?Å), in complex with Mn2+ and citrate (1.9?Å), and with the CDP-DAG substrate (1.8?Å). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism-including a substrate-induced carboxylate shift-for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.
Project description:Solubilization of phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 126.96.36.199) from rat pituitary (GH3) tumours was investigated. PtdIns synthase activity was partially extracted from crude membranes by 3 M-KCl. Prior separation of membranes revealed that a greater proportion of plasma-membrane PtdIns synthase activity was salt-extractable than was endoplasmic reticulum activity. The activity of the salt-extracted enzyme was maximized by low concentrations of 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS; 0.5 mM), Triton X-100 (0.1 mM) or a phospholipid mixture (0.05 mg/ml), but higher concentrations of detergents were inhibitory. The activity of salt-extracted PtdIns synthase was 0.25 +/- 0.08 nmol/min per mg of protein. Salt-extracted PtdIns synthase activity was dependent on Mg2+ (maximal at 0.1 mM) and Mn2+ (maximal at 5 mM), and its pH optimum was in the range 7.0-7.5. The apparent Km for myo-inositol (in the presence of 0.1 mM-CDP-diacylglycerol) was 0.06 mM, and that for CDP-diacylglycerol (at 0.1 mM-myo-inositol) was 0.21 mM. Salt-extracted PtdIns synthase activity was potently inhibited by Ca2+ (50% inhibition at 1 microM), with over 90% inhibition at 10 microM-Ca2+. These data imply the existence of two forms of membrane-associated PtdIns synthase, namely salt-extractable and salt-resistant, with different intracellular localizations. The salt-extractable form of this enzyme may be a useful preparation for further characterization and purification of mammalian PtdIns synthase.
Project description:Myo-inositol is an important constituent of membrane phospholipids and is a precursor for the phosphoinositide signaling pathway. It is synthesized from glucose 6-phosphate by myo-inositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues. It is a putative target for mood-stabilizing drugs such as lithium and valproate. Here, we show that the rat gene (Isyna1) encoding this enzyme generates a number of alternatively spliced transcripts in addition to the fully spliced form that encodes the 68-kDa subunit (the alpha isoform). Specifically, we identify a small 16-kDa subunit (the gamma(c) isoform) derived by an intron retention mechanism and provide evidence for its existence in rat tissues. The gamma(c) isoform is highly conserved in mammals, but it lacks the catalytic domain while retaining the NAD(+) binding domain. Both alpha and gamma(c) isoforms are predominantly expressed in many rat tissues and display apparent stoichiometry in purified enzyme preparations. An IP synthase polyclonal antibody not only detects the alpha and gamma(c) isoforms but also several other isoforms in pancreas, intestine, and testis suggesting that the holoenzyme is composed of unique subunits in various tissues. Interestingly, the alpha isoform is not expressed in the intestine. IP synthase activity assays using purified alpha and gamma(c) isoforms indicate that the latter negatively modulates alpha isoform activity, possibly by competing for NAD(+) molecules. Our findings have important ramifications for understanding the mood stabilization process and suggest that inositol biosynthesis is a highly regulated and dynamic process.
Project description:Diabetes, with only mild ketosis, was induced in male rats by a single injection of streptozotocin. After 12 weeks the specific activities of enzymes concerned with the metabolism of inositol and of inositol lipids were measured in various tissues. Inositol 1-phosphate synthase (EC 188.8.131.52) was most active in testis and the activity was significantly less in diabetic rats than in controls on a similar diet. Inositol oxygenase (EC 184.108.40.206), which converts myo-inositol into glucuronic acid, was also less active in kidney from diabetic animals. CDP-diacylglycerol-inositol phosphatidyltransferase (EC 220.127.116.11) and phosphatidylinositol 4-phosphate kinase (EC 18.104.22.168) showed decreased specific activities in brain and sciatic nerve of diabetic rats. By contrast the diabetic state did not affect the specific activities of phosphatidylinositol kinase (EC 22.214.171.124) or phosphatidylinositol 4,5-bisphosphate phosphatase (EC 126.96.36.199) in these tissues. The results are discussed in relation to diabetic neuropathy.
Project description:Although myo-inositol is included in media for the successful growth of plant tissues, the actual requirement of most tissues, including soybean (Glycine max) callus in suspension culture, for myo-inositol has not been demonstrated. We have made use of deoxyglucose to reduce intracellular levels of myo-inositol. Deoxyglucose is phosphorylated to deoxyglucose 6-phosphate, which inhibits L-myo-inositol 1-phosphate synthase, an important enzyme in the synthesis of myo-inositol. Addition of deoxyglucose to the medium resulted in a decrease in the intracellular level of myo-inositol that corresponded with a decrease in cell division. Cell viability was not affected. When myo-inositol was added to cells along with deoxyglucose, cell division was restored, as were intracellular levels of myo-inositol. Addition of myo-inositol had no affect on the uptake or metabolism of deoxyglucose. From these results we propose that myo-inositol has a role in maintaining cell division in soybean callus tissue in suspension culture.