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Heterologous Over‐expression of α ‐1,6‐Fucosyltransferase from Rhizobium sp.: Application to the Synthesis of the Trisaccharide β ‐ D ‐GlcNAc(1→4)‐ [ α ‐ L ‐Fuc‐(1→6)]‐ D ‐GlcNAc, Study of the Acceptor Specificity and Evaluation of Polyhydroxylated Indolizidines as Inhibitors
Author(s) -
Bastida Agatha,
FernándezMayoralas Alfonso,
Gómez Arrayás Ramón,
Iradier Fatima,
Carretero Juan Carlos,
GarcíaJunceda Eduardo
Publication year - 2001
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/1521-3765(20010601)7:11<2390::aid-chem23900>3.0.co;2-0
Subject(s) - chitobiose , trisaccharide , fucosyltransferase , enzyme , biochemistry , heterologous expression , chemistry , fucose , stereochemistry , recombinant dna , disaccharide , monosaccharide , galactose , gene , chitin , chitosan
An efficient heterologous expression system for overproduction of the enzyme α ‐1,6‐Fucosyltransferase ( α ‐1,6‐FucT) from Rhizobium sp. has been developed. The gene codifying for the α ‐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 α ‐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 α ‐ D ‐GlcNAc‐(1→4)‐[ α ‐ 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 IC 50 of 4.5×10 −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.