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Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability

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Abstract

The effect of metal ions on the activity, the donor substrate specificity, and the stability in organic solvents of Helicobacter pylori α-1,4 fucosyltransferase were studied. The recombinant enzyme was expressed as soluble form in E. coli strain AD494 and purified in a one step affinity chromatography. Its activity was highest in cacodylate buffer at pH 6.5 in the presence of 20 mM Mn2+ ions at 37°C. Mn2+ ions could be substituted by other metal ions. In all cases, Mn2+ ions proofed to be the most effective (Mn2+ > Co2+ > Ca2+ > Mg2+ > Cu2+ > Ni2+ > EDTA). The enzyme shows substrate specificity for Type I disaccharide (1) with a K M of 114 μM. In addition, the H. pylori α-1,4 fucosyltransferase efficiently transfers GDP-activated l-fucose derivatives to Galβ1-3GlcNAc-OR (1). Interestingly, the presence of organic solvents such as DMSO and methanol up to 20% in the reaction medium does not affect significantly the enzyme activity. However, at the same concentration of dioxane, activity is totally abolished.

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Abbreviations

BSA:

Bovine serum albumin

FucT:

Fucosyltransferase

GDP-l-fucose:

Guanosine 5′-diphospho-β-l-fucose

GDP-2-fluoro-l-fucose:

Guanosine 5′-diphospho-2-deoxy-fluoro-β-l-fucose

GDP-d-glucose:

Guanosine 5′-diphospho-β-d-glucose

GDP-l-arabinose:

Guanosine 5′-diphospho-β-l-arabinose

DMSO:

Dimethyl sulfoxide

Lem:

Lemieux spacer, (CH2)8COOMe

Type I:

Galβ-1,3GlcNAc-OR

References

  • Alm RA, Ling SL, Moir DT, King BL, Brown ED, Doig PC, Smith DR, Noonan B, Guild BC, de Jonge BL, Carmel G, Tummino PJ, Caruso A, Uria-Nickelsen M, Mills DM, Ives C, Gibson R, Merberg D, Mills SD, Jiang Q, Taylor DE, Vovis GF, Trust TJ (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176–180

    Article  PubMed  Google Scholar 

  • Appelmelk BJ, Simoons-Smit I, Negrini R, Moran AP, Aspinall GO, Forte JG, De Vries T, Quan H, Verboom T, Maaskant JJ, Ghiara P, Kuipers EJ, Bloemena E, Tadema TM, Townsend RR, Tyagarajan K, Crothers JM, Monteiro MA, Savio A, De Graaff J (1996) Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity. Infect Immun 64:2031–2040

    CAS  PubMed  Google Scholar 

  • Baisch G, Ohrlein R (1997) Convenient chemoenzymatic synthesis of β-purine-diphosphate sugar (GDP-fucose-analogues). Bioorg Med Chem 5:383–391

    Article  CAS  PubMed  Google Scholar 

  • Baisch G, Ohrlein R, Katopodis A, Streiff M, Kolbinger F (1997) Synthetic potential of cloned fucosyltransferase III and VI. Bioorg Med Chem Lett 7:2447–2450

    Article  CAS  Google Scholar 

  • Breton C, Bettler E, Joziasse DH, Geremia RA, Imberty A (1998) Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases. J Biochem (Tokyo) 123:1000–1009

    CAS  Google Scholar 

  • Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, Georgescu AM, Vergez LM, Land ML, Motin VL, Brubaker RR, Fowler J, Hinnebusch J, Marceau M, Medigue C, Simonet M, Chenal-Francisque V, Souza B, Dacheux D, Elliott JM, Derbise A, Hauser LJ, Garcia E (2004) Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci USA 101:13826–13831

    Article  CAS  PubMed  Google Scholar 

  • Chan NW, Stangier K, Sherburne R, Taylor DE, Zhang Y, Dovichi NJ, Palcic MM (1995) The biosynthesis of Lewisx in Helicobacter pylori. Glycobiology 5:683–688

    Article  CAS  PubMed  Google Scholar 

  • Costache M, Apoil PA, Cailleau A, Elmgren A, Larson G, Henry S, Blancher A, Iordachescu D, Oriol R, Mollicone R (1997) Evolution of fucosyltransferase genes in vertebrates. J Biol Chem 272:29721–29728

    Article  CAS  PubMed  Google Scholar 

  • Davies GJ, Gloster TM, Henrissat B (2005) Recent structural insights into the expanding world of carbohydrate-active enzymes. Curr Opin Struct Biol 15:637–645

    Article  CAS  PubMed  Google Scholar 

  • Ernst B, Wagner B, Baisch G, Katopodis A, Winkler T, Oehrlein R (2000) Substrate specificity of fucosyltransferase III: an efficient synthesis of sialyl Lewisx-, Lewisa-derivatives and mimetics thereof. Can J Chem 78:892–903

    Article  CAS  Google Scholar 

  • Gastinel LN, Bignon C, Misra AK, Hindsgaul O, Shaper JH, Joziasse DH (2001) Bovine β1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. EMBO J 20:638–649

    Article  CAS  PubMed  Google Scholar 

  • Ge Z, Chan NW, Palcic MM, Taylor DE (1997) Cloning and heterologous expression of an α1,3-fucosyltransferase gene from the gastric pathogen Helicobacter pylori. J Biol Chem 272:21357–21363

    Article  CAS  PubMed  Google Scholar 

  • Horton JL, Ma B, Palcic MM, Taylor DE (2004) The significance of EXD motifs to the activity of Helicobacter pylori α-1,3/4 fucosyltransferase. Poster presented at the annual conference of the Canadian Society of Microbiology. University of Alberta, Edmonton, pp 20–23

    Google Scholar 

  • Koeller KM, Wong CH (2000) Chemoenzymatic synthesis of sialyl-trimeric-Lewis x. Chemistry 6:1243–1251

    Article  CAS  PubMed  Google Scholar 

  • Kukowska-Latallo JF, Larsen RD, Nair RP, Lowe JB (1990) A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha (1,3/1,4)fucosyltransferase. Genes & Dev 4:1288–1303

    Article  CAS  Google Scholar 

  • Lemieux RU, Bundle DR, Baker DA (1975) The properties of a synthetic antigen related to the human blood-group Lewis a. J Am Chem Soc 97:4076–4083

    Article  CAS  PubMed  Google Scholar 

  • Ma B, Simala-Grant JL, Taylor DE (2006) Fucosylation in prokaryotes and eukaryotes. Glycobiology 16:158–184

    Article  Google Scholar 

  • Martin SL, Edbrooke MR, Hodgman TC, van den Eijnden DH, Bird MI (1997) Lewisx biosynthesis in Helicobacter pylori. Molecular cloning of an α-1,3 fucosyltransferase gene. J Biol Chem 272:21349–21356

    Article  CAS  PubMed  Google Scholar 

  • McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du FY, Hou SF, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK (2001) Complete genome sequence of Salmonella enterica serovar typhimurium LT2. Nature 413:852–856

    Article  CAS  PubMed  Google Scholar 

  • Monteiro MA, Chan KH, Rasko DA, Taylor DE, Zheng PY, Appelmelk BJ, Wirth HP, Yang M, Blaser MJ, Hynes SO, Moran AP, Perry MB (1998) Simultaneous expression of Type I and Type II Lewis blood group antigens by Helicobacter pylori lipopolysaccharides. Molecular mimicry between H. pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms. J Biol Chem 273:11533–11543

    Article  CAS  PubMed  Google Scholar 

  • Murray BW, Takayama S, Schultz J, Wong CH (1996) Mechanism and specificity of human α-1,3 fucosyltransferase V. Biochemistry 35:11183–11195

    Article  CAS  PubMed  Google Scholar 

  • Nunez HA, O’Connor JV, Rosevear PR, Barker R (1981) The synthesis and characterization of α- and β-l-fucopyranosyl phosphates and GDP fucose. Can J Chem 59:2086–2095

    Article  CAS  Google Scholar 

  • Oehrlein R (1999) Glycosyltransferase-catalyzed synthesis of non-natural oligosaccharides. Top Curr Chem 200:227–254

    Article  Google Scholar 

  • Oulmouden A, Wierinckx A, Petit JM, Costache M, Palcic MM, Mollicone R, Oriol R, Julien R (1997) Molecular cloning and expression of a bovine α-1,3 fucosyltransferase gene homologous to a putative ancestor gene of the human FUT3-FUT5-FUT6 cluster. J Biol Chem 272:8764–8773

    Article  CAS  PubMed  Google Scholar 

  • Palma S, Morais VA, Coelho AV, Costa J (2004) Effect of the manganese ion on human alpha-3/4 fucosyltransferase III activity. Biometals 17:35–43

    Article  CAS  PubMed  Google Scholar 

  • Qasba PK, Ramakrishnan B, Boeggeman E (2005) Substrate-induced conformational changes in glycosyltranferases. Trends Biochem Sci 30:53–62

    Article  CAS  PubMed  Google Scholar 

  • Rabbani S, Miksa V, Wipf B, Ernst B (2005a) Molecular cloning and functional expression of a novel Helicobacter pylori alpha-1, 4 fucosyltransferase. Glycobiology 15:1076–1083

    Article  CAS  PubMed  Google Scholar 

  • Rabbani S, Compostella F, Franchini L, Wagner B, Panza L, Ernst B (2005b) Synthetic potential of fucosyltransferase III for the synthesis of fluorescent-labeled milk oligosaccharides. J Carbohyd Chem 24:789–807

    Article  CAS  Google Scholar 

  • Rasko DA, Wang G, Palcic MM, Taylor DE (2000) Cloning and characterization of the α-1,3/4 fucosyltransferase of Helicobacter pylori. J Biol Chem 275:4988–4994

    Article  CAS  PubMed  Google Scholar 

  • Shao J, Li M, Jia Q, Lu Y, Wang PG (2003) Sequence of Escherichia coli O128 antigen biosynthesis cluster and functional identification of an α-1,2 fucosyltransferase. FEBS Lett 553:99–103

    Article  CAS  PubMed  Google Scholar 

  • Shinoda K, Morishita Y, Sasaki K, Matsuda Y, Takahashi I, Nishi T (1997) Enzymatic characterization of human α-1, 3 fucosyltransferase Fuc-TVII synthesized in a B cell lymphoma cell line. J Biol Chem 272:31992–31997

    Article  CAS  PubMed  Google Scholar 

  • Sun HY, Lin SW, Ko TP, Pan JF, Liu CL, Lin CN, Wang AH, Lin CH (2007) Structure and mechanism of Helicobacter pylori fucosyltransferase: a basis for lipopolysaccharide variation and inhibitor design. J Biol Chem 282:9973–9982

    Article  CAS  PubMed  Google Scholar 

  • Tomb JF, White O, Clayton KerlavageAR, RA SuttonGG, Fleischmann RD, Ketchum KA, Klenk HP, Gill S, Dougherty BA, Nelson K, Quackenbush J, Zhou L, Kirkness EF, Peterson S, Loftus B, Richardson D, Dodson R, Khalak HG, Glodek A, McKenney K, Fitzegerald LM, Lee N, Adams MD, Hickey EK, Berg DE, Gocayne JD, Utterback TR, Peterson JD, Kelley JM, Karp PD, Smith HO, Fraser CM, Venter JC (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539–547

    Article  CAS  PubMed  Google Scholar 

  • Ünligil UM, Zhou S, Yuwaraj S, Sarkar M, Schachter H, Rini JM (2000) X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J 19:5269–5280

    Article  PubMed  Google Scholar 

  • Wang G, Boulton PG, Chan NW, Palcic MM, Taylor DE (1999) Novel Helicobacter pylori alpha-1,2 fucosyltransferase, a key enzyme in the synthesis of Lewis antigens. Microbiology 145:3245–3253

    CAS  PubMed  Google Scholar 

  • Wang G, Ge Z, Rasko DA, Taylor DE (2000) Lewis antigens in Helicobacter pylori: biosynthesis and phase variation. Mol Microbiol 36:1187–1196

    Article  CAS  PubMed  Google Scholar 

  • Wiggins CAR, Munro S (1998) Activity of the yeast MNN1α-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc Natl Acad Sci USA 95:7945–7950

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Financial support was generously provided by the Swiss National Science Foundation. In addition, we thank YAMASA Corp., Tokyo/Japan for the generous gift of GDP-l-fucose.

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  1. Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland

    Said Rabbani, Francesco Corona & Beat Ernst

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  1. Said Rabbani
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  2. Francesco Corona
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  3. Beat Ernst
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Correspondence to Beat Ernst.

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Rabbani, S., Corona, F. & Ernst, B. Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability. Biometals 22, 1011–1017 (2009). https://doi.org/10.1007/s10534-009-9252-1

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  • DOI: https://doi.org/10.1007/s10534-009-9252-1

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