Heterologous Over-expression ofα-1,6-Fucosyltransferase fromRhizobium sp.: Application to the Synthesis of the Trisaccharideβ-D-GlcNAc(1→4)- [α-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.

Preparation and Characterization of a Bifunctional Aldolase/Kinase Enzyme: A More Efficient Biocatalyst for CC Bond Formation

“…After that, the culture was centrifuged at 3,000 x g during 30 min at 4 ºC and the resulting pellet was treated with lysozyme and DNase for protein extraction. [41] The recombinant protein containing an N-terminal 6xHis tag was purified in a Ni +2 -IDAagarose column pre-equilibrated with sodium phosphate buffer (20 mM, pH 7.5). FBPA was eluted with the same buffer containing imidazole 1 M. All the fractions containing protein were pooled together and further purified by size-exclusion chromatography on a HiLoad 26/60 Superdex 75 PG column controlled using the AKTA-FPLC system (GE Healthcare Life Science).…”

In Vivo Chaperone‐Assisted Folding of α‐1,6‐Fucosyltransferase from Rhizobium sp.

“…in E. coli. 8 In this heterologous system the enzyme was mainly expressed as IBs. Several attempts to refold the recombinant enzyme in vitro were unsuccessful.…”

Indolizidine and quinolizidine alkaloids

“…Ref. 25 Several unnatural deoxy analogues of castanospermine synthesised during the review period include (±)-6-deoxycastanospermine 47, 26 and (ϩ)-1-deoxycastanospermine 48 and (ϩ)-1-deoxy-8a-epi-castanospermine 49 from a derivative of -glucose. 17 The X-ray crystal structure of 1,7,8-tri-O-acetyl-6-O-benzoylcastanospermine 41 has been determined, and shows chair and twist conformations for the six-and five-membered rings, respectively.…”