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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Membrane fractions from a lon strain of Escherichia coli but not a wild-type strain catalyze the incorporation of fucose from guanosine 5'-diphosphate-fucose into a lipid and into polymeric material. Both incorporation reactions specifically require only uridine 5'-diphosphate (UDP)-glucose. The sugar lipid was shown to be an intermediate in the synthesis of the polymer which was related to colanic acid. The sugar lipid had the structure (fucose3, glucose2)-glucose P-P-lipid. Its behavior on column and thin-layer chromatography, the rates of its hydrolysis in acid and base, and the response of its synthesis to inhibitors are all identical to the other sugar-lipid intermediates which have been shown to contain sugars attached to the C55-polyisoprenol, undecaprenol, by a pyrophosphate linkage. The membrane fractions from both the lon strain and the wild-type strain also catalyzed the incorporation of either glucose from UDP-glucose or galactose from UDP-galactose into a lipid fraction which was shown to contain the free sugar attached by a monophosphate linkage to an undecaprenol-like lipid. This lipid was isolated and its nuclear magnetic resonance spectra was identical to undecaprenol. The membrane fractions from both strains also incorporated glucose from UDP-glucose into glycogen and into a polymer that behaved like Escherichia coli lipopolysaccharide. Conditions were found where the incorporation of glucose could be directed specifically into each compound by adding the appropriate inhibitors.
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PMID:Role of a sugar-lipid intermediate in colanic acid synthesis by Escherichia coli. 31 40

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.
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PMID:Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5. 32 11

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.
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PMID:Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase. 225 16

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.
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PMID:Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens. 230 61

The role of sfrB and rfaH genes in the regulation of expression of membrane glycosyltransferases was studied in Escherichia coli and Salmonella typhimurium. The transferase enzymes form part of a multienzyme system involved in biosynthesis of the polysaccharide core of Gram-negative bacterial lipopolysaccharides. Several sfrB mutants of E. coli showed reductions of 90-98% in the activities of two of the glycosyltransferases (UDP-galactose:(glucosyl)lipopolysaccharide 1,6-galactosyltransferase and UDP-glucose: (glucosyl)lipopolysaccharide 1,3-glucosyltransferase). Introduction of a recombinant ColE1 plasmid restored the transferase levels to normal and simultaneously corrected the F-factor defects that also characterize sfrB mutants; recombinant plasmids containing other regions of the E. coli chromosome were ineffective. An amber mutation of the S. typhimurium rfaH gene (thought to be the homologue of the E. coli sfrB gene) resulted in 97% loss of activity of the Salmonella UDP-galactose:(glucosyl)lipopolysaccharide galactosyltransferase. Antibody precipitation studies showed that the loss of enzyme activity in the amber mutant was associated with a corresponding decrease in amount, but not in size, of the transferase protein, indicating that the gene is not the structural gene for the S. typhimurium galactosyltransferase. Taken together, the results indicate that the sfrB(rfaH) gene acts as a positive regulatory element in expression of multiple glycosyltransferases in E. coli and S. typhimurium.
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PMID:Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium. 623 Mar 55

The incorporation of rhamnose and glucose into the core part of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa was studied using enzyme preparations from strain PAC1R and LPS-defective mutants derived from it. Crude membrane preparations from the LPS-defective mutant PAC556 transferred rhamnose from dTDP-L-[14C]rhamnose to material insoluble in trichloracetic acid. The preparations contained both transferase enzyme and acceptor, the former being destroyed by heating. Between 60 and 70% of the radioactive rhamnose transferred to the membranes was extractable by aqueous phenol and non-diffusible. The material extracted did not move in any of the chromatography solvents tested and contained rhamnose as the sole radioactive component. Soluble dTDP-L-rhamnose-LPS rhamnosyltransferase was obtained from the parent strain PAC1R by ammonium sulphate precipitation of a 105000 g supernatant fraction from broken bacteria. It was most active at pH 8 with 5 mM-MgCl2 and required heat-treated membranes of PAC556 as acceptor. This mutant, whose LPS lacks both O-antigenic side-chains and rhamnose in the core, was shown to lack either the epimerase or the NADP-dependent oxidoreductase used to synthesize dTDPrhamnose. After preincubation with soluble transferase and UDPglucose, heated membranes of mutant strains PAC611, PAC612 and PAC605 could also act as acceptors for rhamnose. These mutants all lacked some or all of the glucose as well as the rhamnose from the core of their LPS and the experiments thus provided confirmation that rhamnose was the terminal hexose of the core in P. aeruginosa PAC1.
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PMID:Biosynthesis of the core part of the lipopolysaccharide of Pseudomonas aeruginosa. 677 64

A Drosophila UDP-glucose:glycoprotein glucosyltransferase was isolated, cloned and characterized. Its 1548 amino acid sequence begins with a signal peptide, lacks any putative transmembrane domains and terminates in a potential endoplasmic reticulum retrieval signal, HGEL. The soluble, 170 kDa glycoprotein occurs throughout Drosophila embryos, in microsomes of highly secretory Drosophila Kc cells and in small amounts in cell culture media. The isolated enzyme transfers [14C]glucose from UDP-[14C]Glc to several purified extracellular matrix glycoproteins (laminin, peroxidasin and glutactin) made by these cells, and to bovine thyroglobulin. These proteins must be denatured to accept glucose, which is bound at endoglycosidase H-sensitive sites. The unusual ability to discriminate between malfolded and native glycoproteins is shared by the rat liver homologue, previously described by A.J. Parodi and coworkers. The amino acid sequence presented differs from most glycosyltransferases. There is weak, though significant, similarity with a few bacterial lipopolysaccharide glycotransferases and a yeast protein Kre5p. In contrast, the 56-68% amino acid identities with partial sequences from genome projects of Caenorhabditis elegans, rice and Arabidopsis suggest widespread homologues of the enzyme. This glucosyltransferase fits previously proposed hypotheses for an endoplasmic reticular sensor of the state of folding of newly made glycoproteins.
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PMID:Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins. 772 8

Serum resistance of gonococci in most patients is due to sialylation of a Gal beta 1-4GlcNAc group on a conserved 4.5 kDa lipopolysaccharide (LPS) component by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) catalysed by a gonococcal sialyl transferase. This sialylation is enhanced by a low M(r) factor(s) which, like CMP-NANA, is released in diffusates from high M(r) fractions obtained from sonicates dialysed at 4 degrees C. Also, as shown here, this factor(s) is released when the sonicates are dialysed at 18-20 degrees C. The enhancement of sialylation, first demonstrated using enzymes in gonococcal extracts, has been shown to occur in live gonococci and hence probably to have a role in pathogenicity. Gonococci, emerging from lag phase and incubated for 2 h with CMP-14CNANA fixed up to 90% more radiolabel than controls when the second factor(s) was present; their LPS separated by SDS-PAGE contained more radiolabel than control samples and label was not detected in any other component. Fractions with enhancing activity absorbed maximally at about 260 nm but a mixture of UDP-galactose (UDP-Gal), UDP-N-Acetyl galactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc) and UDP-N-Acetyl glucosamine (UDP-GlcNAc) showed no significant enhancing activity. The enhancing action of the low M(r) fractions was unaffected by incubation with beta-galactosidase.
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PMID:Sialylation of lipopolysaccharide by CMP-NANA in viable gonococci is enhanced by low Mr material released from blood cell extracts but not by some UDP sugars. 783 May 28

The biosynthetic function of the lgtABE genetic locus of Neisseria meningitidis was determined by structural analysis of lipopolysaccharide (LPS) derived from mutant strains and enzymic assay for glycosyltransferase activity. LPS was obtained from mutants generated by insertion of antibiotic resistance cassets in each of the three genes lgtA, lgtB, lgtE of the N. meningitidis immunotype L3 strain phi3 MC58. LPS from the parent strain expresses the terminal lacto-N-neotetraose structure, Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc. Mild hydrazine treatment of the LPS afforded O-deacylated samples that were analyzed directly by electrospray ionization mass spectrometry (ESI-MS) in the negative ion mode. In conjunction with results from sugar analysis, ESI-MS revealed successive loss of the sugars Gal, GlcNAc, and Gal in lgt B, lgt A, and lgt E LPS, respectively. The structure of a sample of O- and N-deacylated LPS derived by aqueous KOH treatment of lgt B LPS was determined in detail by two-dimensional homo- and heteronuclear NMR methods. Using a synthetic beta-GlcNAc acceptor and a beta-lactose acceptor, the glycosyltransferase activities encoded by the lgtB and lgtA genes were unambiguously established. These data provide the first definitive evidence that the three genes encode the respective glycosyltransferases required for biosynthesis of the terminal trisaccharide moiety of the lacto-N-neotetraose structure in Neisseria LPS. From ESI-MS data, it was also determined that the Gal-deficient LPS expressed by the lgt E mutant is identical to that of the major component expressed by immunotype L3 galE-deficient strains. The galE gene which encodes for UDP-glucose-4-epimerase plays an essential role in the incorporation of Gal into meningococcal LPS.
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PMID:Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. 870 94

Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.
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PMID:Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i. 944 88


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