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Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.

ABSTRACT: Lewis X (Le(x))-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Le(x) and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5'-diphosphate-beta-l-fucose (GDP-fucose), the universal fucosyl donor, the Le(x) trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits l-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts l-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori alpha1,3 fucosyltransferase, we prepared a library of Le(x) trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Le(x)-mediated biological processes.

SUBMITTER: Wang W

PROVIDER: S-EPMC2752511 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.

Wang Wei W Hu Tianshun T Frantom Patrick A PA Zheng Tianqing T Gerwe Brian B Del Amo David Soriano DS Garret Sarah S Seidel Ronald D RD Wu Peng P

Proceedings of the National Academy of Sciences of the United States of America 20090904 38

Lewis X (Le(x))-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Le(x) and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5'-diphosphate-beta-l-fucose (GDP-fucose), the universal fucosyl donor, the Le(x) trisaccharide, and their C-5 substituted derivatives ca ...[more]

PMID: 19805264

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Project description:Rhizobial NodZ ?1,6-fucosyltransferase (?1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-?-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two ?1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of ?1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35?Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98?Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among ?1,2-, ?1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand ?C2 and helix ?C3. In addition, there is a shift of the ?C3 helix itself upon GDP-Fuc binding.

Dataset Information

Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.

Publications

Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.

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