Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.     

Structure of the O-polysaccharide of Providencia stuartii O4 containing 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O4 was obtained by mild acid degradation of the lipopolysaccharide, and the following structure of the pentasaccharide repeating unit was established: [structure: see text] where D-Qui4N(L-AspAc) is 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose, which has not been hitherto found in bacterial polysaccharides. Structural studies were performed using sugar and methylation analyses, Smith degradation and NMR spectroscopy, including conventional 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments as well as COSY and NOESY experiments run in an H(2)O-D(2)O mixture to reveal correlations for NH protons.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of P. aeruginosa O:3a, b and O:3a, d lipopolysaccharides

On mild acid degradation of Pseudomonas aeruginosa O:3a,b and O:3a,d lipopolysaccharides O-specific polysaccharides were isolated. Both polysaccharides were found to contain 2-acetamido-2,6-dideoxy-D-galactose, identified as fucosamine hydrochloride formed after hydrolysis with a very low yield. The other two components of the trisaccharide repeating unit, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, were identified without isolation in their free state directly in the course of structural investigation of the polysaccharides. Both these monosaccharides have never before been found in nature. Solvolysis of either O:3a,b or O:3a,d polysaccharides with liquid hydrogen fluoride resulted in the formation of the same trisaccharide, N-acetylfucosamine residue being the reducing end. The structure of this trisaccharide, which is the repeating unit of both polysaccharides, was deduced from the results of successive chemical modifications and 13C-nuclear magnetic resonance spectra recorded for every oligosaccharide formed. As a result, the acidic diaminosugars were converted into 2,3-diacetamido-2,3-dideoxy-D-mannose indistinguishable from authentic sample. The O-specific polysaccharides O:3a,b and O:3a,d differed in the configuration of the glycosidic bond of N-acetylfucosamine residue only and had the following structures: leads to 4)DManImU(beta 1 leads to 4)DMan(NAc)2U (beta 1 leads to 3)DFucNAc(beta 1- leads to 4)DManImU(beta 1 leads to 4)DMan(NAc)2U (beta 1 leads to 3)DFucNAc(alpha 1- where DManImU = 2.3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2, 3-dideoxy-D-mannuronic acid, DMan(NAc)2U = 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, DFucNAc = 2-acetamido-2,6-dideoxy-D-galactose. The structures established were in agreement with optical rotations and assignments of all the signals in the 13C-nuclear magnetic resonance spectra of the polysaccharides.     

Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1 --> 3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-[N-[(R)-3-hydroxybutyryl]-L-alanyl]amino-D-glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: --> 4)-beta-D-GlcpA-(1 --> 3)-beta-D-GlcpNAc-(1 --> 2)-beta-D-Quip4N(HbAla)-(1 --> 3)-alpha-D-Galp-(1 -->. This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

New structure for the O-polysaccharide of Providencia alcalifaciens O27 and revised structure for the O-polysaccharide of Providencia stuartii O43

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O27 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. It was found that the polysaccharide is built up of linear partially O-acetylated tetrasaccharide repeating units and has the following structure: [structure: see text] where Qui4NFo stands for 4-formamido-4,6-dideoxyglucose (4-formamido-4-deoxyquinovose). The O-polysaccharide structure of Providencia stuartii O43 established earlier was revised with respect to the configuration of the constituent 4-amino-4,6-dideoxyhexose (from Rha4N to Qui4N).     

Extracellular polysaccharides of Erwinia futululu,a bacterium associated with a fungal canker disease of Eucalyptus spp

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-[(R)-1-carboxyethylidene)-Galp. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O1 (Lányi), O3 (Habs), O13 and O14 (Wokatsch), and the serologically related strain NCTC 8505

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).     

N-(1-Carboxyethyl)alanine (alanopine), a new non-sugar component of lipopolysaccharides of Providencia and Proteus

O-Polysaccharides were released by mild acid degradation of lipopolysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76 and were studied by 1D and 2D ¹H and ¹³C NMR spectroscopies, including HMBC and NOESY (ROESY) experiments. Both polysaccharides were found to contain N-(1-carboxyethyl)alanine (alanopine) that is N-linked to 4-amino-4,6-dideoxyglucose. Analysis of published data [Vinogradov, E.; Perry, M. B. Eur. J. Biochem.2000, 267, 2439-2446] shows that alanopine is present also on the same sugar in the lipopolysaccharide core of Proteus mirabilis O6 and O57.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lányi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.     

Structure and characterization of the gene cluster of the O-antigen of <Emphasis Type="Italic">Escherichia coli</Emphasis> O49 containing 4,6-dideoxy-4-[(<Emphasis Type="Italic">S</Emphasis>-3-hydroxybutanoylamino]-D-glucose

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of enteropathogenic Escherichia coli O49 and studied by sugar analysis along with one- and two-dimensional 1H- and 13C-NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit of the O-polysaccharide was established: [formula], where D-Qui4N(S3HOBut) stands for 4,6-dideoxy-4-[(S)-3-hydroxybutanoylamino]-D-glucose and O-acetylation of GlcNAc is partial (~30%). To our knowledge, no N-(3-hydroxybutanoyl) derivative of Qui4N has been hitherto found in bacterial polysaccharides. Gene functions of the O-antigen gene cluster of E. coli O49 were assigned by bioinformatics analysis and found to correspond to the O-polysaccharide structure. Two new genes were revealed and suggested to be responsible for synthesis and transfer of the 3-hydroxybutanoyl group.     

Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA. The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharides of E. coli O126. It was found that, amongst the monosaccharides, β- d -N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure α- D -Gal p (1 → 3)-β- D -Glc pNAc(1→2)- D -Man p.     

Structure of a fucoidan from the brown seaweed Fucus serratus L

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and galactose were isolated from the brown seaweed Fucus serratus L. The fucoidan structure was investigated by 1D and 2D 1H and 13C NMR spectroscopy of its desulfated and de-O-acetylated derivatives as well as by methylation analysis of the native and desulfated polysaccharides. A branched structure was suggested for the fucoidan with a backbone of alternating 3- and 4-linked alpha-L-fucopyranose residues, -->3)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->, about half of the 3-linked residues being substituted at C-4 by trifucoside units alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-alpha-L-Fucp-(1-->. Minor chains built up of 4-linked alpha-fucopyranose and beta-xylose residues were also detected, but their location, as well as the position of galactose residues, remained unknown. Sulfate groups were shown to occupy mainly C-2 and sometimes C-4, although 3,4-diglycosylated and some terminal fucose residues may be nonsulfated. Acetate was found to occupy C-4 of 3-linked Fuc and C-3 of 4-linked Fuc in a ratio of about 7:3.     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.     

Investigation of substrate water interactions at the high-affinity Mn site in the photosystem II oxygen-evolving complex

18 O isotope exchange measurements of photosystem II (PSII) in thylakoids from wild-type and mutant Synechocystis have been performed to investigate binding of substrate water to the high-affinity Mn4 site in the oxygen-evolving complex (OEC). The mutants investigated were D1-D170H, a mutation of a direct ligand to the Mn4 ion, and D1-D61N, a mutation in the second coordination sphere. The substrate water 18 O exchange rates for D61N were found to be 0.16+/-0.02 s(-1) and 3.03+/-0.32 s(-1) for the slow and fast phases of exchange, respectively, compared with 0.47+/-0.04 s(-1) and 19.7+/-1.3 s(-1) for the wild-type. The D1-D170H rates were found to be 0.70+/-0.16 s(-1) and 24.4+/-4.6 s(-1) and thus are almost within the error limits for the wild-type rates. The results from the D1-D170H mutant indicate that the high-affinity Mn4 site does not directly bind to the substrate water molecule in slow exchange, but the binding of non-substrate water to this Mn ion cannot be excluded. The results from the D61N mutation show an interaction with both substrate water molecules, which could be an indication that D61 is involved in a hydrogen bonding network with the substrate water. Our results provide limitations as to where the two substrate water molecules bind in the OEC of PSII.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structures of the O1B and O1C lipopolysaccharide antigens of Escherichia coli.

The O-specific moieties of the O1B antigen (lipopolysaccharide) from Escherichia coli O1B:K1 and the O1C antigen from E. coli O1C:K- both consist of L-rhamnose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in a molar ratio of 2:1:1:1. By using fragmentation procedures, methylation analysis, and one- and two-dimensional nuclear magnetic resonance spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In the O1B polysaccharide X is 2, and in the O1C polysaccharide X is 3. With the recently published structure of the O1A polysaccharides (B. Jann, A. S. Shashkov, D. S. Gupta, S. M. Panasenko, and K. Jann, Carbohydr. Polym. 18:51-57 1992), three related O1 antigens are now known. Their common (O1-specific) epitope is suggested to be the side-chain N-acetyl-D-mannosamine residue.