Structure and Function of Helicobacter pylori Fucosyltransferase
Date Issued
2007
Date
2007
Author(s)
Lin, Sheng-Wei
DOI
en-US
Abstract
Helicobacter pylori is well known as the primary cause of gastritis, duodenal
ulcers, and gastric cancer. The lipopolysaccharide (LPS) of this pathogen contains
Lewis x and Lewis y structures in the terminus to mimic the surface carbohydrates
of gastric epithelial cells, which is proposed to escape the surveillance of host
immune system. H. pylori α1,3-fucosyltransferase catalyzes the fucose transfer
from the donor GDP-fucose to the acceptor N-acetyllactosamine (LacNAc). The
research progress was previously hampered by either poor protein expression or the
marginal solubility of the protein. The work in the thesis at first greatly improved
the marginal solubility of the full-length protein by systematic deletion of the C
terminus of H. pylori α1,3-FucT, which made it possible for further investigations.
Based on the biophysical characterizations, including CD spectroscopy and
analytical ultracentrifugation, up to 80 residues, including the tail rich in positive
and hydrophobic residues (sequence 434-478) and half of the ten heptad repeats
(399-433), can be removed without significant change in structure and catalysis.
Half of the heptad repeats are required to maintain both secondary and native
quaternary structures (dimeric form). Removal of more residues in the C terminus
led to major structural alteration, which was correlated with the loss of enzymatic
activity. In accordance with the thermal denaturation studies, the results support the
idea that a higher number of tandem repeats, functioning to facilitate a dimeric
structure, helps to prevent the protein from unfolding when incubated at higher
temperatures.
H. pylori α1,3-FucT was also subjected to biochemical characterizations,
such as substrate specificity, specific chemical modifications, and site-directed
mutagenesis. H. pylori α1,3-FucT is highly specific to the β1,4-linkage and does
tolerate modification in the reducing end. Both sugar residues of LacNAc are
essential for activity. H. pylori α1,3-FucT can sterically accommodate an additional
sugar introduced at either C2 or C3 of galactose, but not C4 and C6 at the same
sugar. Furthermore, when oligomeric LacNAc was subjected to the enzymatic
fucosylation, one to several fucose residues observed in the mass spectra. Every
LacNAc unit can be fucosylated in a random manner. H. pylori α1,3-FucT is
sensitive to the modification of diethylpyrocarbonate (His-specific) and
phenylglyoxal (Arg), but not N-ethylmaleimide (Cys), indicating that His and Arg
play an important role in the reaction. The site-directed mutagenesis study showed
that Arg79, Arg89, Arg118, and Arg354 are mainly involved in the acceptor
binding, while Arg79 and Arg195 are essential for the binding with both substrates.
Although cysteine residues do not participate in the catalysis, an intramolecular
disulfide linkage between Cys159 and Cys237 are determined. The other residues,
Cys168 and Cys353, have free thiol in their side chains.
Additionally, protein crystallization was successful with the C-terminal
deletion of 115 residues. Three crystal structures have been solved, including the
enzyme/GDP-fucose complex and the enzyme/GDP complex. The structure is
composed of two Rossmann-like fold domains, typical of the glycosyltransferase-B
(GT-B) family. Specific interactions with GDP and GDP-fucose bound to the active
site induce conformational changes in the C-terminal domain. Structural
comparison with other GT-B members suggests Glu95 to serve as the general base
in catalysis. The residues Arg195, Tyr246, Glu249 and Lys250 function to interact
with the donor substrate. Asn240 is involved in the binding with the acceptor.
Glu249 is proposed to stabilize the developing oxonium cation during the reaction.
Although the crystallized protein lacks a substantial C terminus, these structures not
only reveal subunit interactions for a dimeric structure, but also predict the location
of the missing heptad repeats. We propose a catalytic mechanism and a model of
polysaccharide binding to explain the observed variations in H. pylori LPS, as well
as to facilitate the development of potent inhibitors. Taken together, this thesis
provides clear understanding for the structure and function relationship of H. pylori
α1,3-FucT.
Subjects
幽門螺旋桿菌
岩藻醣轉移酶
Helicobacter pylori
Fucosyltransferase
SDGs
Type
other
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