Study the active site of Helicobactor pylori α-1,3-fucosyltransferase by site-directed mutagenesis
Date Issued
2006
Date
2006
Author(s)
Liu, Chia-Ling
DOI
zh-TW
Abstract
Helicobacter pylori (H. pylori) is considered to be a primary cause of gastritis, duodenal ulcer and gastric cancer. This pathogenic bacterium produces Lex and Ley epitopes in the O-antigens of lipopolysaccharides (LPS) to mimic the carbohydrate antigens on the surface of gastric epithelial cells. The molecular mimicry avoids the detection by the host immune system. The enzyme α-1,3-fucosyltransferase (α1,3-FucT) from H. pylori catalyzes the glycosyl addition of fucose from the donor GDP-fucose to the acceptor LacNAc. It is a membrane associated protein. Previously, our lab carried out a systematic truncation of the C terminus of the α-1,3-FucT to improve the protein solubility. C-Terminal 45 amino acid residues include the amphiphilic helices that anchor the protein to the cell membrane. The corresponding protein, C45-FucT, was water soluble and had the same activity as that of the full-length enzyme. Next to the amphiphilic helices is the heptad repeats. Deletion of 115 residues removed both helices and the heptad repeats in the C terminal end. Because heptad repeats were expected to form the leucine zipper and facilitated the formaitnon of a dimeric structure, therefore C115-FucT was found to be a monomer with low activity.
Due to lack of structural imformation of fucosyltransferase, it is not easy to understand the catalytic mechanism. In order to study the residues involed in catalysis and substrate binding, this thesis lays a special emphasis on His and Arg. The methods include the use of modifiers and preparation of different mutant proteins by site-directed mutagenesis.
DEPC (diethylpyrocarbonate) and PGO (phenylglycoxal) were shown to specifically react with His and Arg, respectively. Both of these modifier can inactivate the α-1,3-FucT of H. pylori. When α-1,3-FucT was incubated with GDP-Fuc before the addition of PGO or DEPC, the higher concentration of GDP-Fuc was used in the study, the higher level of activity was retained. The protection effect of GDP-Fuc indicated that His and Arg were likely located at either the active site or the binding site of the donor substrate.
Ten Arg residues and ten His residues were individually mutated to Ala and the resulting mutants were analyzed by the activity assay. The activity assay was a coupling assay carried out based on the production of the side product GDP. The result indicated the mutants R79A, R118A, R180A, R195A and R354A were completely inactive. R89A, H60A, H131A, H282A, H298A and H345A were found to have reduced activities. The Km and Vmax of these mutants were also measured. According to Km, H282, H298 and H354 were found to be involved in the binding with GDP-Fuc, Whereas H60 and R89 were related to the binding with LacNAc.
The structure of was solved by x-ray crystallograpgy in the collaboration with Dr. Andrew Wang’s group of this institute. So far only C115-FucT can be crystallized for prove the structural imformation. The results suggest that R195, N240, E249, K250 directly interact with GDP-Fuc. In contrast, the kinetic analysis supports the idea that N240 is involved in the binding with LacNAc. The superimposition of C115-FucT to the structure of BGT (β-Glucosyltransferase) revealed that E95 may act as the catalytic base in general catalytic base. We also try to figure out the real structure of the C45-FucT.
Subjects
幽門螺旋桿菌
岩藻榶轉移酶
Helicobacter pylori
fucosyltransferase
SDGs
Type
other
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