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Escherichia coli O86 possesses high human blood group B activity because of its O-antigen structure, sharing the human blood group B epitope. In this study, the wbwK gene of E. coli O86:B7 was expressed and purified as the GST fusion protein. Thereafter, the wbwK gene was biochemically identified to encode an α1,2-fucosyltransferase through radioactivity assays, as well as mass spectrometry and NMR spectroscopy. WbwK shows strict substrate specificity and only recognizes Galβ1,3GalNAcα-OR (T-antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen. In contrast to other α1,2-fucosyltransferases, WbwK does not display activity toward the simple substrate Galβ-OMe. Comparison with another recently characterized α1,2-fucosyltransferase (WbsJ) of E. coli O128:B12 indicates a low level of amino acid identity between them; however, they share a common acceptor substrate, Galβ1,3GalNAcα-OR. Domain swapping between WbwK and WbsJ revealed that the smaller variable domains located in the C-terminus determine substrate specificity, whereas the larger variable domain in the N-terminus might play a role in forming the correct conformation for substrate binding or for localization of the α1,2-fucosyltransferase involved in O-antigen biosynthesis. In addition, milligram scale biosynthesis of the H-type 3 blood group antigen was explored using purified recombinant WbwK. WbwK may have potential applications in masking T-antigen, the tumor antigen, in vivo

biosynthesis, gene, O-antigen, Escherichia coli, epitope, Substrate Specificity, T-Antigen, human blood group B

NCBI PubMed ID: 18842005
Publication DOI: 10.1021/bi801067s
Journal NLM ID: 0370623
Publisher: American Chemical Society
Correspondence: csc@chem.wayne.edu; wang.892@osu.edu
Institutions: Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202, and Department of Biochemistry, The Ohio State UniVersity, Columbus, Ohio 43210
Methods: 13C NMR, 1H NMR, ESI-MS, NMR-1D, serological methods, genetic methods, biochemical methods

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-β-L-fucose synthetase) to GDP-β-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-β-L-fucose was optimized and the final product was purified. The formation of GDP-β-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-β-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-β-L-fucose

enzymatic synthesis, GDP-β-L-fucose, 6-deoxy-hexose, GDP-D-mannose 4, 6-dehydratase, GDP-4-keto-6-deoxy-D-mannose 3, 5-epimerase-4-reductase (=GDP-β-L-fucose synthetase)

NCBI PubMed ID: 10988249
Publication DOI: 10.1093/glycob/10.9.875
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Institutions: Chemische Mikrobiologie, Bergische Universitat Wuppertal, Germany
Methods: 13C NMR, 1H NMR, SDS-PAGE, 31P NMR, genetic methods, biochemical methods, HPLC

After the breakthroughs in genomic sequencing, one of the next challenges remains to understand the molecular biology of other classes of biomolecules, such as protein and lipids, many of which carry specific glycomodification when mediating their biological functions. This review focuses on the 6-deoxyhexose biosynthesis of cell surface glycans of three Gram-negative pathogens, Helicobacter pylori, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans serotype a. 6-Deoxysugars are important functional components of cell surface glycans, and their biosynthetic pathways might be suitable targets for novel interventions of antibacterial chemotherapy.

rhamnose, fucose, nucleotide sugars, 6-deoxy-talose

NCBI PubMed ID: 14693916
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Correspondence: risto.renkonen@helsinki.fi
Institutions: Rational Drug Design Program and Department of Bacteriology and Immunology, Biomedicum and Haartman Institute, PO Box 63, FIN-00014 University of Helsinki, Helsinki, Finland