'Wave-type' structure of a synthetic hexaglycosylated decapeptide: a part of the extracellular domain of human glycophorin A.

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy-alpha-D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with phi angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.

Hydride-Meisenheimer complex formation and protonation as key reactions of 2,4,6-trinitrophenol biodegradation by Rhodococcus erythropolis.

Biodegradation of 2,4,6-trinitrophenol (picric acid) by Rhodococcus erythropolis HLPM-1 proceeds via initial hydrogenation of the aromatic ring system. Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic sigma-complex) of picric acid and its protonated form under physiological conditions. These complexes are key intermediates of denitration and productive microbial degradation of picric acid. For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time. Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid. This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite. Cell extracts of R. erythropolis HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol. Picric acid is used as the sole carbon, nitrogen, and energy source by R. erythropolis HLPM-1.

Structural studies on the lipopolysaccharide from a rough strain of Ochrobactrum anthropi containing a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide lipid A backbone.

A degradation protocol using de-O-acylation and subsequent alkaline de-N-acylation was applied to the lipopolysaccharide of Ochrobactrum anthropi rough strain LMG 3301. Three main oligosaccharide bisphosphates containing core-lipid A backbone structures were obtained after fractionation by anion-exchange HPLC. Using 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, and NOE spectroscopy (ROESY and NOESY), the following structures were established: [formula: see text] where Kdo is 3-deoxy-D-manno-octulosonic acid, D-GlcN3N is 2,3-diamino-2,3-dideoxy-D- glucose and R is H or alpha-D-GalpA or 4-deoxy-beta-L-threo-hex-4-enopyranuronic acid, the latter sugar being derived from alpha-D-GalpA by beta-elimination of a substituent attached to 0-4. This is the first report on the isolation from a lipopolysaccharide of an oligosaccharide containing GlcN3N in the lipid A backbone [beta-D-GlcpN3N4P-(1-->6)-alpha-D-GlcpN3N1 P]. Sugar and methylation analysis confirmed the presence of the GalA-->Kdo disaccharide and non-stoichiometric substitution of GalA. It is suggested that Glc is the substituent at 0-4 in GalA and that in the non-degraded lipopolysaccharide the amino group of GlcN is not acylated.

Identification and synthesis of new bicyclic acetals from the mountain pine beetle, Dendroctonus ponderosae Hopkins (Col.: Scol.).

Head-space volatiles obtained from male mountain pine beetles, Dendroctonus ponderosae, were analyzed by coupled GC-MS and chiral gas chromatography. 5-Ethyl-7-methyl-6,8-dioxabicyclo[3.2.1]octane (6) was found as a new naturally occurring isomer of brevicomin (1). In addition, several stereoisomers of 7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octan-2-ol (11) and 1-(5-methyl-6,8-dioxabicyclo[3.2.1]octyl)ethanol (12) could be identified. Relative and absolute configurations of the compounds were determined by unambiguous syntheses, which are described.

Biotransformation of diphenyl ether by the yeast Trichosporon beigelii SBUG 752.

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.

Preferred conformations and dynamics of five core structures of mucin type O-glycans determined by NMR spectroscopy and force field calculations.

Glycosyltransferases acting on O-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes and O-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Gal beta (1-3)GalNAc alpha Bzl (1, core 1), GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha Bzl (2, core 2), GlcNAc beta (1-3)GalNAc alpha Bzl (3, core 3), GlcNAc beta (1-6)[GlcNAc beta (1-3)]GalNAc alpha Bzl (4, core 4), GlcNAc beta (1-6)GalNAc alpha Bzl (5, core 6), the elongated core 2, Gal beta (1-4)GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha pNp (6) and GalNAc alpha-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1-6) linkage is much more flexible than the (1-3) or the (1-4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additional N-acetyl group in (3). The conformational equilibrium of beta (1-3) glycosidic bonds of 1 and 3 was not affected by attaching a beta (1-6) linked GlcNAc unit to the GalNAc residue in 2 and 4. However, experimental and theoretical data show that the beta (1-6) linkages of the trisaccharides 2 and 4, which carry an additional beta (1-3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1-6) linkage of 2, 4, and 6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of approximately 4 kcal mol-1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.

Chemical structure of the carbohydrate backbone of Vibrio parahaemolyticus serotype 012 lipopolysaccharide.

The chemical structure of the saccharide portion of Vibrio parahaemolyticus serotype 012 lipopolysaccharide was studied. Using chemical degradation and modification, as well as methylation analysis in combination with GLC-MS, laser-desorption mass spectrometry and 1H-NMR and 13C-NMR spectroscopy, the carbohydrate backbone of the lipopolysaccharide was characterized as a branched decasaccharide with the following structure: (formula; see text) In the native lipopolysaccharide two additional phosphate groups are present and 3-deoxy-D-threo-hexulosonic acid and D-galacturonic acid are bound via acid-labile linkages.

[Conformational analysis of N-terminal O-glycopeptide sequences of interleukin-2]. / Konformationsanalytische Untersuchungen von N-terminalen O-glycopeptidsequenzen des Interleukin-2.

The preferred conformations of eight O-glycopeptide sequences from the N-terminus of interleukin-2 containing two to ten amino acids, monoglycosylated at Thr3 with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, were determined by means of n.m.r. spectroscopic methods. The preferred conformation of the N-terminal sequence, L-Ala-L-Pro-[alpha-D-GalpNAc-(1----3)]-L-Thr-L-Ser, including the O-glycosidically linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group is not substantially influenced by the linkage of additional amino acids at the C-terminal end. Extended conformations were observed for all peptide units. Measurements of the relaxation times of the 13C atoms showed that the 2-acetamido-2-deoxy-D-galactose bound to the central amino acids has the lowest mobility, whereas the terminal amino acid residues and peptide side-chains are flexible. Calculations with the force-field program AMBER yielded conformations of minimized energies that were in good agreement with the n.m.r. spectroscopic data. This was only true when n.m.r. parameters that can be used as starting values for the calculations were available. Comparison with a nonglycosylated, N-terminal tetrapeptide sequence analog did not suggest changes in the peptide conformation when Thr3 is glycosylated with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group.

[Conformation analysis of D-xylose-containing O-glycopeptide sequences]. / Konformationsanalytische Untersuchungen von D-Xylose-haltigen O-Glycopeptidsequenzen.

The preferred conformations of the sequences of O-glycopeptides containing a beta-D-xylosyl group linked to an L-seryl residue, found in the N-terminus of proteodermatan sulfate, show an almost extended peptide chain with the sugar unit in a specific position. The results of force-field calculations with the AMBER program have been confirmed, by n.m.r.-spectroscopic experiments, for a minimum conformation.

Transformation of 3-chlorodibenzofuran by Pseudomonas sp. HH69.

The dibenzofuran-degrading bacterial strain Pseudomonas sp. HH69 showed high oxidative activity towards 3-chlorodibenzofuran (3CDF). During the co-metabolic turnover of 3CDF large amounts of 4-chlorosalicylate and temporarily small amounts of salicylate were excreted. Simultaneously a yellow colour appeared due to the excretion of two polar products. Conversion of 3CDF by a mutant, derived from Pseudomonas sp. HH69 and defective in 2,3-dihydroxybiphenyl-1,2-dioxygenase led to the formation of equal quantities of 4'-chloro-2,2',3-trihydroxybiphenyl (4'CTHBP) and 4-chloro-2,2',3-trihydroxybiphenyl (4CTHBP). Crude extracts of the wild type transformed 4'CTHBP to 4-chlorosalicylate, whilst 4CTHBP was transformed to salicylate. Hence, we propose a non-selective initial attack on both aromatic rings of 3CDF and a degradative pathway for the resulting chlorotrihydroxybiphenyls.

Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27.

A Pseudomonas sp. strain, HH69, and a mixed culture, designated HH27, were isolated by selective enrichment from soil samples. The pure strain and the mixed culture grew aerobically on dibenzofuran as the sole source of carbon and energy. Degradation proceeded via salicylic acid which was branched into the gentisic acid and the catechol pathway. Both salicylic acid and gentisic acid accumulated in the culture medium of strain HH69. The acids were slowly metabolized after growth ceased. The enzymes responsible for their metabolism showed relatively low activities. Besides the above-mentioned acids, 2-hydroxyacetophenone, benzopyran-4-one (chromone), several 2-substituted chroman-4-ones, and traces of the four isomeric monohydroxydiben-zofurans were identified in the culture medium. 2,2',3-Trihydroxybiphenyl was isolated from the medium of a dibenzofuran-converting mutant derived from parent strain HH69, which can no longer grow on dibenzofuran. This gives evidence for a novel type of dioxygenases responsible for the attack on the biarylether structure of the dibenzofuran molecule. A meta-fission mechanism for cleavage of the dihydroxylated aromatic nucleus of 2,2',3-trihydroxybiphenyl is suggested as the next enzymatic step in the degradative pathway.

Transformation of Dibenzo-p-Dioxin by Pseudomonas sp. Strain HH69.

Dibenzo-p-dioxin was oxidatively cleaved by the dibenzofuran-degrading bacterium Pseudomonas sp. strain HH69 to produce minor amounts of 1-hydroxydibenzo-p-dioxin and catechol, while a 2-phenoxy derivative of muconic acid was formed as the major product. Upon acidic methylation, the latter yielded the dimethylester of cis, trans-2-(2-hydroxyphenoxy)-muconic acid.

Isolation and chemical analysis of 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose as a constituent of the lipopolysaccharides of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli and Vibrio cholerae.

Methyl 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-hepto+ ++ pyranoside (1) was released from the lipopolysaccharide of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli by methanolysis and isolated by high-voltage paper electrophoresis. Its chemical structure was determined by chemical analysis, deamination with nitrous acid, g.1.c.-m.s., and 1H- and 13C-n.m.r. spectroscopy performed on its acetylated derivative. The disaccharide moiety of 1 was also detected in lipopolysaccharides of Vibrio cholerae.

Isolation and structural characterization of an 8-O-(4-amino-4-deoxy-beta-L-arabinopyranosyl)-3-deoxy-D-manno- octulosonic acid disaccharide in the lipopolysaccharide of a Proteus mirabilis deep rough mutant.

On short treatment of the lipopolysaccharide of a deep rough mutant of Proteus mirabilis (strain R45) with hydrochloric acid (0.01 M, 10 min, 100 degrees C) a compound was released which, on high-voltage paper electrophoresis, migrated slightly to the cathode (MGlcN = 0.19) and which stained with ninhydrin, alkaline silver nitrate and in the thiobarbituric acid assay. After purification and derivatization, its chemical structure was identified by combined gas-liquid chromatography/mass spectrometry and two-dimensional shift-correlated proton nuclear magnetic resonance spectroscopy as 8-O-(4-amino-4-deoxy-beta-L-arabinopyranosyl)-3-deoxy-D-manno-octu losonic acid. This disaccharide was also present in a wild-type strain of P. mirabilis.

[Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond]. / Konformationsanalyse modifizierter Tetrasaccharidsequenzen vom Typ der N-Glycoproteine--zum Problem der alpha-(1----6)-glycosidischen Bindung.

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.

[Conformational analysis of the double pentasaccharide sequence of the "bisected" structure ov N-glycoproteins]. / Konformationsanalyse der verzweigten Pentasaccharid-sequenz der "bisected" Struktur von N-Glycoproteinen.

In the conformational analysis of the pentasaccharide alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D- Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcNAc, which represents the essential core structure within the bisected type of N-glycoproteins, n.m.r. experiments and GESA calculations indicated that a gg conformation is preferred for the (1----6)-glycosidic linkage. This is in contrast to the results obtained for the tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D- Manp-(1----4)- D-GlcNAc, which indicated that a gt conformation with the (1----6)-linked alpha-D-mannose group folded back is preferred. The conformations of the remaining glycosidic linkages in the tetra- and penta-saccharide are similar.