Biosynthesis of isoprenoids via mevalonate in Archaea: the lost pathway.
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Isoprenoid compounds are ubiquitous in living species and diverse in biological function. Isoprenoid side chains of the membrane lipids are biochemical markers distinguishing archaea from the rest of living forms. The mevalonate pathway of isoprenoid biosynthesis has been defined completely in yeast, while the alternative, deoxy-D-xylulose phosphate synthase pathway is found in many bacteria. In archaea, some enzymes of the mevalonate pathway are found, but the orthologs of three yeast proteins, accounting for the route from phosphomevalonate to geranyl pyrophosphate, are missing, as are the enzymes from the alternative pathway. To understand the evolution of isoprenoid biosynthesis, as well as the mechanism of lipid biosynthesis in archaea, sequence motifs in the known enzymes of the two pathways of isoprenoid biosynthesis were analyzed. New sequence relationships were detected, including similarities between diphosphomevalonate decarboxylase and kinases of the galactokinase superfamily, between the metazoan phosphomevalonate kinase and the nucleoside monophosphate kinase superfamily, and between isopentenyl pyrophosphate isomerases and MutT pyrophosphohydrolases. Based on these findings, orphan members of the galactokinase, nucleoside monophosphate kinase, and pyrophosphohydrolase families in archaeal genomes were evaluated as candidate enzymes for the three missing steps. Alternative methods of finding these missing links were explored, including physical linkage of open reading frames and patterns of ortholog distribution in different species. Combining these approaches resulted in the generation of a short list of 13 candidate genes for the three missing functions in archaea, whose participation in isoprenoid biosynthesis is amenable to biochemical and genetic investigation. (+info)
The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum.
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The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man(5)GlcNAc(2)-PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man(3)GlcNAc(2)-PP-dolichol and Man(4)GlcNAc(2)-PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif. (+info)
Anomeric configuration of N-acetylglucosaminyl phosphorylundecaprenols formed in Bacillus cereus Membranes.
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The structural difference was studied between two N-acetylglucosaminyl phosphorylundecaprenols formed by incubation of Bacillus cereus membranes with UDP-N-acetylglucosamine. On the treatment with 50% phenol, the major one of these glycolipids (Lipid 1) yielded a saccharide phosphate, while the other (Lipid 2) yielded N-acetylglucosamine along with a saccharide phosphate. The saccharide phosphates from Lipids 1 and 2 were identified as alpha-N-acetylglucosamine 1-phosphate and its beta-anomer, respectively, based on their susceptibility to acid, alpha- and beta-N-acetylglucosaminidases, and UDP-N-acetylglucosamine pyrophosphorylase. Thus, it seems most probable that Lipids 1 and 2 were alpha- and beta-N-acetylglucosaminyl phosphorylundecaprenols, respectively. (+info)
Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein.
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The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical. (+info)
Microbial isoprenoid biosynthesis and human gammadelta T cell activation.
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Human Vgamma9/Vdelta2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non-peptide antigens has been enigmatic until recently. The break-through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) (HMB-PP), turned out to be by far the most potent Vgamma9/Vdelta2 T cell activator known, with an EC(50) of 0.1 nM. (+info)
Salmonella serogroups C2 and C3 identified by agglutination using an immunoglobulin G3(kappa) monoclonal antibody (32-1-E3) reactive with a somatic factor 8-like polysaccharide antigen.
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An immunoglobulin G3(kappa) monoclonal antibody (MAb), MAb 32-1-E3, which was prepared in BALB/c mice by using a heated, alcohol-acetone-extracted Salmonella newport CDC 50 antigen, reacted with protein-free lipopolysaccharides from Salmonella groups C2 (O:6,8) and C3 (O:-,8) but not with those from any other serogroup tested. Sodium periodate did not inhibit antigen reactivity, which was consistent with its identity as the abequose-containing disaccharide O:8 antigen. Reactivity was inhibited by competition with serogroup C2 (O:6,8) and C3 (O:-,8) antigens but not with non-O:8 antigens. Reactivity was also inhibited by preincubation of the antigen with polyclonal rabbit antiserogroup C2 or C3 antibodies but not with antisera to serogroup C1 or other Salmonella serogroups. The MAb agglutinated with all strains of Salmonella serogroups C2 and C3 tested but not with other bacteria. Agglutination was inhibited by preabsorbing the MAb with either of two serogroup C3 Salmonella strains, S. virginia CDC 189 or S. haardt MDL 83A4545, which contain only O:8, but not by preabsortion with O:8-negative S. cholerasuis MDL 81A7623 (group C1; O:6,7), S. paratyphi type B CDC 157 (group B; O:1,[4],5,12), or Escherichia coli (O:157) (which contains no Salmonella serogroup antigens). The MAb reacted strongly (4+ agglutination) with all 140 wild-type strains of group C2 and C3 Salmonella spp. tested and showed no reaction with any of 1,324 wild-type strains of non-C2 or non-C3 Salmonella spp. tested. The MAb is useful as a replacement for absorbed, polyclonal, single-factor O:8 antiserum to discriminate Salmonella serogroups C2 and C3 from serogroup C1. (+info)
Formation of unusual mannosamine-containing lipid-linked oligosaccharides in Madin-Darby canine kidney cell cultures.
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Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is the major lipid-linked oligosaccharide (LLO) produced by Madin-Darby canine kidney cells in culture. However, when these cells are incubated in the presence of millimolar concentrations of mannosamine and labeled with [2-3H]mannose, they accumulate various LLO that have smaller-sized oligosaccharides with unusual structures and the Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol is not detected. Thus in the presence of 10 mM mannosamine, more than 80% of the oligosaccharides are eluted from concanavalin A-Sepharose with 10 mM alpha-methylglucoside, indicating that they no longer have the tight-binding characteristics of control oligosaccharides. In addition, 20-40% of these oligosaccharides bind to Dowex 50-H+, indicating the presence of mannosamine in these structures. Interestingly enough, these abnormal oligosaccharides are still transferred to protein. The mannosamine-induced oligosaccharides were separated into neutral and basic fractions on a cation exchange resin. The neutral oligosaccharides ranged in size from hexose3(GlcNAc)2 to hexose10(GlcNAc)2 with the major species being Man5(GlcNAc)2 to Man7(GlcNAc)2. These oligosaccharides were almost completely susceptible to digestion by alpha-mannosidase and by endoglucosaminidase H. The basic oligosaccharides showed anomolous behavior on the Bio-Gel P-4 columns and appeared to be of small size on the standard columns, ranging from hexose2 to hexose4. However, most of these oligosaccharides were susceptible to digestion by endoglucosaminidase H as well as by alpha-mannosidase, suggesting that they were of different size and structure than would be predicted from the gel filtration patterns. Significantly, when the basic oligosaccharides were subjected to chemical N-acetylation, or when the gel filtration columns were run at high pH rather than at the usual pH of 3.0, the basic oligosaccharides migrated like much larger oligosaccharides. These data provide strong evidence to indicate that some mannosamine can be incorporated into the LLO, and that these mannosamine-containing oligosaccharides exhibit unusual properties. Preliminary studies indicated that Madin-Darby canine kidney cells do incorporate label from [3H]mannosamine into the LLO. (+info)
Teaching dolichol-linked oligosaccharides more tricks with alternatives to metabolic radiolabeling.
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The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed. (+info)