title: Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors.
abstract: An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.
authors.affiliation: Departamento de Química Orgánica Biológica, Instituto de Química Orgánica General, CSIC, Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Bastida
authors.forename: A
authors.initials: A
authors.lastname: Fernández-Mayoralas
authors.forename: R
authors.initials: R
authors.lastname: Gómez Arrayás
authors.forename: F
authors.initials: F
authors.lastname: Iradier
authors.forename: J C
authors.initials: JC
authors.lastname: Carretero
authors.forename: E
authors.initials: E
authors.lastname: García-Junceda
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D001426:Bacterial Proteins; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007212:Indolizines / Q000378:metabolism / Q000494:pharmacology; D008279:Magnetic Resonance Imaging; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D017026:Swainsonine / Q000378:metabolism; D014312:Trisaccharides / Q000138:chemical synthesis*; D054836:Indolizidines
chemical_list: D000118:Acetylglucosaminidase; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005647:Fucosyltransferases; D007212:Indolizines; D011994:Recombinant Proteins; D017026:Swainsonine; D014312:Trisaccharides; C103941:NodZ protein, bacteria; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; C037806:castanospermine; D054836:Indolizidines