Abstract
alpha 1,3-Fucosyltransferase (FucT) from H. pylori catalyzes the transfer of fucose from the donor GDP-beta-fucose in alpha 1,3-linkage to the acceptor beta-Gal-1,4-beta-GlcNAc (LacNAc) to produce the Lewis X trisaccharide. Lewis antigens are mainly expressed in the O-antigen of the H. pylori lipopolysaccharide and structurally similar to tumor-associated carbohydrate antigen found in the host. The enzyme contains a C-terminal heptad repeats that function as leucine zipper to facilitate the formation of a dimeric structure. In this thesis work, protein crystallization became successful only with the deletion of C-terminal 115 residues. We solved three crystal structures, including the protein, the protein-substrate (GDP-fucose) complex, and the protein-product (GDP) complex. The solved structures indicate that the enzyme is composed of two Rossmann-like fold domains, typical of the GT-B family of glycosyltransferases. Specific interactions with GDP and GDP-fucose bound to the active site induced conformational changes in the C-terminal domain. Structural comparison with other GT-B members suggested that Glu95 in the N-terminal domain plays the role of general base in catalysis, as confirmed by site-directed mutagenesis. Other mutants at Arg195, Asn240, Glu249 and Lys250 also showed significant decrease in the enzymatic activity. EDTA treatment showed that FucT does not require divalent metal ion. Based on these observations, a catalytic mechanism was proposed. Besides, the truncated FucT formed a dimer in crystal, which may characterize the full-length enzyme.
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