Biosynthesis of heparin. Hydrogen exchange at carbon 5 of the glycuronosyl residues

Prihar, Harry S.; Campbell, Patrick; Feingold, David S.; Jacobsson, Ingvar; Jensen, John W.; Lindahl, Ulf; Rodén, Lennart

doi:10.1021/bi00544a016

Cited by 39 publications

(13 citation statements)

References 21 publications

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“…However, the mechanism proposed for this epimerization is different than that proposed for non-uronic acids. A microsomal enzyme known as heparosan-N-sulfate D-glucuronosyl 5-epimerase involved in the biosynthesis of heparin has been studied in detail, and the reaction was proposed to proceed via a carbanion intermediate at C-5 (30,31). Using tritiated substrates, the possibility of a mechanism involving a hexos-4-eenuronosyl intermediate or a hexos-3-eenuronosyl intermediate was eliminated because the label was only lost from the C-5 position (30).…”

Section: Table I Nmr Analysis Of Compounds Udp-␤-l-pnenac and Udp-␤-lmentioning

confidence: 99%

“…A microsomal enzyme known as heparosan-N-sulfate D-glucuronosyl 5-epimerase involved in the biosynthesis of heparin has been studied in detail, and the reaction was proposed to proceed via a carbanion intermediate at C-5 (30,31). Using tritiated substrates, the possibility of a mechanism involving a hexos-4-eenuronosyl intermediate or a hexos-3-eenuronosyl intermediate was eliminated because the label was only lost from the C-5 position (30). Since the product of the WbjB reaction (UDP-2-acetamido-2,6-dideoxy-␤-L-arabino-4-hexulose), which is the substrate for the WbjC reaction contains a keto group, both the C-5 and C-3 epimerization reactions most likely occur via a mechanism similar to that of RmlC.…”

Section: Table I Nmr Analysis Of Compounds Udp-␤-l-pnenac and Udp-␤-lmentioning

confidence: 99%

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Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

Mulrooney

Poon

McNally

et al. 2005

Journal of Biological Chemistry

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UDP-N-acetyl-L-fucosamine is a precursor to L-fucoThose results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-L-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.

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Section: Table I Nmr Analysis Of Compounds Udp-␤-l-pnenac and Udp-␤-lmentioning

confidence: 99%

Section: Table I Nmr Analysis Of Compounds Udp-␤-l-pnenac and Udp-␤-lmentioning

confidence: 99%

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

Mulrooney

Poon

McNally

et al. 2005

Journal of Biological Chemistry

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“…The repeating structure of the hyaluronic acid from group A streptococci is the 4-~-glucuronyl-1,3-~-N-acetylglucosaminyl unit [48] and the teichuronic acid of Bacillus suhtilis is the 4-glucuronyl-1,3-N-acetylgalactosaminyl unit [49,SO]. Just as the K5 antigen is reminiscent of the precursor polysaccharide of heparin [43,44], that of the teichuronic acid is reminiscent of the desulfo-chondroitin sulfates A and C. In none of these aminosugar-containing bacterial polysaccharides L-iduronic acid was found. The 5-epimerization is dependent on sulfation of the amino and hydroxyl groups [43,44], reaction5 which d o not seem to occur in bacteria.…”

Section: F~~g M E~f U~i O N Of the Cnrboxyl-reduced Po1ysacchuride Bmentioning

confidence: 99%

“…Just as the K5 antigen is reminiscent of the precursor polysaccharide of heparin [43,44], that of the teichuronic acid is reminiscent of the desulfo-chondroitin sulfates A and C. In none of these aminosugar-containing bacterial polysaccharides L-iduronic acid was found. The 5-epimerization is dependent on sulfation of the amino and hydroxyl groups [43,44], reaction5 which d o not seem to occur in bacteria.…”

Section: F~~g M E~f U~i O N Of the Cnrboxyl-reduced Po1ysacchuride Bmentioning

confidence: 99%

The Structure of the Capsular Polysaccharide (K5 Antigenn) of Urinary-Tract-Infective Escherichia coli 010:K5:H4. A Polymer Similar to Desulfo-Heparin

et al. 1981

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The capsular polysaccharide was isolated from Escherichia coli 010 : K5 : H4; it could not be obtained from a uncapsulated ( K Y ) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1 : 1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-fi-glucuronyl-I ,4-a-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.It has been shown previously that pathogenicity of Escherichia coli bacteria correlated with the chemical and physical properties of their surface polysaccharide antigens [I, 21. E. coli strains with certain capsular polysaccharide antigens (K antigens) are frequently associated with urinary tract infections, especially in children [2 -41. The K antigens which are most prominent in these extraintestinal infections are KI, K2, K3, K5, K12 and K13. Encapsulated bacteria give rise to antibodies against K antigens in man and experimental animals. However, antibodies against the K5 polysaccharide antigen are only very rarely found after immunization with E. coli exhibiting the K5 antigen. It was therefore desirable to elucidate the structure of the K5 antigen in the hope that structural considerations may help to explain the poor immunogenicity of this important bacterial surface antigen.The following structural information of the above-mentioned K antigenic capsular polysaccharides of E. coli is available : the K1 antigen is a homopolysaccharide consisting of cc-2,S-linked neuraminic acid [5], the K2 antigen is a teichoicacid-like polymer consisting of galactopyranosyl-glycerol phosphate and galactofuranosyl-glycerol phosphate units in a molar ratio of 2: 1 [6] and the K13 antigen is a heteropolysaccharide with a repeating sequence of 3-linked ribose and 7-linked 2-keto-3-deoxy-manno-octulosonic acid (dOclA) [7]. In preliminary studies we have found that the K12 antigen consists of L-rhamnose and dOclA in a molar ratio of 2: 1 (unpublished results). In this publication we report the structure of the KS antigen from E. coli 010 : K5 : H4, compare it with similar bacterial polysaccharides and discuss the low immunogenicity of this capsular polysaccharide. MATERIALS AND METHODS Bacteria and CultivationEscherichia coli strain Bi 8337-41 (010 : K5 : H4) was used. It was obtained by Drs I. and F. Qrskov of the International Abbreviation. dOclA, 2-kelo-3-deoxy-~-manno-octulosonic acid Enzyme. b-Glucuronidase (EC 3.2.1.31).Escherichia Centre, Copenhagen. The growth conditions for the isolation of the capsular polysaccharide have been described previously [6,7]. Isolation and Purijication qf the Capsular PolysaccharideThe acidic capsular polysaccharide and the bacterial cells were precipitated from the liquid cultures by the additi...

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“…The C-5 epimerase involved in the biosynthesis of heparin\HS has been purified from bovine liver [10] and cloned from a bovine lung cDNA library [11]. The epimerase reaction occurs by the abstraction and re-addition of the C-5 proton via a carbanion intermediate with an inversion of configuration such that the carboxy group is shifted across the plane of the pyranose ring [12][13][14]. In a solubilized enzyme system the reaction is freely reversible, and incubations performed in the presence of $H # O result in the incorporation of $H into both IdoA and GlcA units (see Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5

Hagner-McWhirter

Lindahl

2000

Biochemical Journal

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In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAβ1-4GlcNSO3α1-]n), or of O-desulphated heparin (predominantly [4IdoAα1-4GlcNSO3α1-]n) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of 3H2O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of 3H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-3H-labelled polysaccharide products showed that the 3H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].

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Biosynthesis of heparin. Hydrogen exchange at carbon 5 of the glycuronosyl residues

Cited by 39 publications

References 21 publications

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

The Structure of the Capsular Polysaccharide (K5 Antigenn) of Urinary-Tract-Infective Escherichia coli 010:K5:H4. A Polymer Similar to Desulfo-Heparin

Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5

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