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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.

AuthorsBastida, Agatha CSIC ORCID ; Fernández-Mayoralas, Alfonso CSIC ORCID ; Gómez Arrayás, Ramón; Iradier, Fátima; Carretero, Juan Carlos; García-Junceda, Eduardo CSIC ORCID
Keywordsenzyme alpha 1,6-Fucosyltransferase
Rhizobium sp
Trisaccharide beta-D-GlcNAc(1-4)-[alpha-L-Fuc-(1-6)]-D-GlcNAc
Polyhydroxylated Indolizidines
Issue Date2001
CitationChemistry 2001, 7, 2390-2397
AbstractAn 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 amplyfied 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 5 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 beta-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 know the structural requirements for acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides structuraly related to chitobiose. The enzyme is able to use, among others, the disaccharide N-acetyl lactosamine (4) as a good substrate and the monosaccharide GlcNAc (8) as a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potencial inhibitors of the enzyme. The indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10-5 M and, interestingly, is the one that could better mimic the structural features of the fucose moiety in the presumed transition state.
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