title: Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability.
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 KM 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.
authors: Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors: Said
authors: S
authors: Rabbani
authors: Francesco
authors: F
authors: Corona
authors: Beat
authors: B
authors: Ernst
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D004187:Disaccharides; D008670:Metals; D011994:Recombinant Proteins; D012997:Solvents; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases