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

An efficient heterologous expression system fur overproduction of the enzym e alpha -1,6-Fucosyltransferase (alpha -1,6-FucT) from Rhizobium sp, has be en developed. The gene codifying for the alpha -1.6-FucT was amplified by P CR using specific primers. After purification, the gene was cloned in the p lasmid pKK2233. 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 increas e 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 pre paration of the recombinant enzyme was used for the synthesis of the branch ed trisaccharide alpha -D-GlcNAc-(1 --> 4)-[alpha -L-Fuc-(1 --> 6)]-D-GlcNA c (3), from chitobiose (2) and GDP-Fucose (1). After purification, the tris accharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme wa s studied towards mono-, di- and trisaccharides, which are structurally rel ated to chitobiose. The enzyme uses, among others, the disaccharide N-acety l lactosamine as a good substrate: the monosaccharide GlcNAc is a weak acce ptor. Finally, several racemic polyhydroxylated indolizidines have been tes ted as potential inhibitors of the enzyme. Indolizidine 21 was the best inh ibitor 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 i n the presumed transition state.