Enzymatic synthesis and structure of two glycolipids from Type XIV Pneumococcus

Kaufman, Bernard; Kundig, F. Dodyk; Distler, Jack J.; Roseman, Saul

doi:10.1016/0006-291x(65)90705-9

Cited by 45 publications

(19 citation statements)

References 9 publications

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“…2 (lane 3-6) shows that the overexpressed CpoA (spDGS) was able to catalyze the glycosylation of only ␣-MGlcDAG by using the sugar nucleotide UDP-Gal. This is in agreement with earlier characterization of the lipid composition in S. pneumoniae, where the two major glycolipids were characterized to be monoglucosyl diacylglycerol (MGlcDAG) and galactosyl-glucosyldiacylglycerol (GalGlcDAG) (36,37), and where all sugars were in ␣-configuration and identically linked as in A. laidlawii (38,39). We have recently characterized the ORF5 gene adjacent downstream to cpoA as encoding the S. pneumoniae ␣-MGlcDAG glucosyltransferase (spMGS) (6).…”

Section: Structural Features Of Bacterial Lipid Glycosyltransferasessupporting

confidence: 91%

Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids inAcholeplasma laidlawii and Streptococcus pneumoniae

Edman¹,

Berg²,

Storm

et al. 2003

Journal of Biological Chemistry

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In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) ␣-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC 2.4.1.157) and the (ii) ␣-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N-and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N-and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

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Section: Structural Features Of Bacterial Lipid Glycosyltransferasessupporting

confidence: 91%

Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids inAcholeplasma laidlawii and Streptococcus pneumoniae

Edman¹,

Berg²,

Storm

et al. 2003

Journal of Biological Chemistry

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“…A diglucosyl diglyceride has been isolated from Staphylococcus aureus 11341 and identified in several other species of Staphylococcus and in B. subtilis [35]. Galactosylglucosyl diglyceride has been shown to occur in Pneumococcus [36,37], 8. faecalis [24] and Lactobacillus cmei [35]. M .…”

Section: Results and Discussiopu'mentioning

confidence: 99%

The Chemical Composition of the Cytoplasmic Membrane of Bacillus subtilis

Bishop¹,

Rutberg

Samuelsson³

1967

European Journal of Biochemistry

136

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The cytoplasmic membrane of Bacillus subtilis 168, prepared from cells in the stationary Analyses of the membrane lipid revealed the presence of phospholipids (75OiO), neutral lipid and a compound identified as a diglucosyl diglyceride. The major phospholipids were diphosphatidyl glycerol and phosphatidyl ethanolamine, with small amounts of phosphatidyl glycerol and lipoamino acids.Branched chain fatty acids comprised over 7501, of the total fatty acids of both whole cells and membranes. Is0 and anteiso acids with 15 and 17 carbon atoms were the major components, together with small amounts of is0 acids containing 14 and 16 carbon atoms and n-acids. No unsaturated acids were present. phase, has been found to contain protein (62O/,), RNA (22O/,) and lipid (160/0). [4,5] take place a t the membrane.Although the cytoplasmic membrane holds a central position in bacterial metabolism, the detailed composition of the membrane has been studied in relatively few species. Whilst protein and lipid are apparently ubiquitous constituents of the membrane, the amount of RNA and carbohydrate present has been found to vary widely. Many of these variations can no doubt be ascribed to differences in species and culture conditions, but the lack of uniformity in the analyses makes it desirable that further information be obtained.In order to provide a basis for a detailed investigation of membrane structure in Bacillus subtilis, the chemical composition of the membrane has been determined. MATERIALS AND METHODS Organism and Growth ConditionsBacillus subtilis strain 168 was used throughout. Cells were grown in a medium containing (per litre), NH4C1, 2 g ; K,HPO,, 14 g ; KH,PO,, 6 g ; Na citrate 2 H,O, 1 g; Difco casamino acids, 10 g; pH, 7.6. After autoclaving, MgSO,, 0.2 g and glucose, 5 g were added together with trace metals. An overnight Extraction of the casamino acids used in the culture medium by refluxing with chloroform showed that the fatty acid content was less than 0.5 mg per 100 g of casamino acids. Preparation of Protoplasts and MembranesThe cells were suspended in 0.2 M phosphate buffer pH 6.6 containing 0.25 M sucrose, a t a concentration of 1-2 x loll cells/ml. 1.3 mg of lysozyme and 2 pg of pancreatic DNAase were added per ml of suspension and the cells incubated at 30" for 30 to 60 min until microscopic examination showed > 9Q0/, protoplast formation. The protoplasts were collected by centrifugation (40,000 x g for 50 min) and ruptured by resuspension in distilled water containing mM Mg++ and DNAase (1 pg/ml). The mixture was incubated at room temperature until the viscosity decreased to a level where the solution could be readily pipetted. The membranes were collected by centrifugation (48,000 x g for 30 min), washed five times with 0.Qo/, (w/v) NaCl and stored frozen a t Reagents Lysozyme was obtained from the Sigma Chemical Company and DNAase from Worthington Biochemical Corporation. Analytical grade solvents were used as purchased, all other solvents were redistilled before use.-15".

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“…However, this lipid anchor has not been detected in pneumococcal membranes (14), and there are still no explanations as to how it becomes a part of LTA. In contrast, according to model B, the required lipid anchor is Glc(␣133)-acyl 2 Gro, which is known to be a major glycolipid of pneumococcal membranes (6,14,27).…”

Section: Discussionmentioning

confidence: 99%

“…To investigate these problems, we hypothesized that the repeating unit begins with AATGal instead of ribitol and that the repeating unit is anchored to Glc(␣133)-acyl 2 Gro (Fig. 1B), which is abundantly present in the pneumococcal membrane (6,14,27). Our evaluation of the two proposed models of LTA structure (models A and B) follows.…”

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confidence: 99%

A New Model of Pneumococcal Lipoteichoic Acid Structure Resolves Biochemical, Biosynthetic, and Serologic Inconsistencies of the Current Model

Seo¹,

Cartee²,

Pritchard

et al. 2008

J Bacteriol

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Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(␤133)AATGal(␤133)Glc(␣133)-acyl 2 Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(␣133)GalNAc(␤13, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(␣133)-acyl 2 Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.

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Enzymatic synthesis and structure of two glycolipids from Type XIV Pneumococcus

Cited by 45 publications

References 9 publications

Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids inAcholeplasma laidlawii and Streptococcus pneumoniae

Structural Features of Glycosyltransferases Synthesizing Major Bilayer and Nonbilayer-prone Membrane Lipids inAcholeplasma laidlawii and Streptococcus pneumoniae

The Chemical Composition of the Cytoplasmic Membrane of Bacillus subtilis

A New Model of Pneumococcal Lipoteichoic Acid Structure Resolves Biochemical, Biosynthetic, and Serologic Inconsistencies of the Current Model

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