Biochemical characterization of Helicobacter pylori α-1,4 fucosyltransferase: metal ion requirement, donor substrate specificity and organic solvent stability.
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.
Rabbani
Said
S
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Corona
Francesco
F
Ernst
Beat
B
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
D001426:Bacterial Proteins; D002412:Cations; D004187:Disaccharides; D008670:Metals; D011994:Recombinant Proteins; D012997:Solvents; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases