 |
PDBsum entry 3bub
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Probing the substrate specificity of golgi alpha-Mannosidase ii by use of synthetic oligosaccharides and a catalytic nucleophile mutant.
|
|
|
Authors
|
|
W.Zhong,
D.A.Kuntz,
B.Ember,
H.Singh,
K.W.Moremen,
D.R.Rose,
G.J.Boons.
|
|
|
Ref.
|
|
J Am Chem Soc, 2008,
130,
8975-8983.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan
processing pathway, provides a route to blocking cancer-induced changes in cell
surface oligosaccharide structures. To probe the substrate requirements of GMII,
oligosaccharides were synthesized that contained an alpha(1,3)- or
alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not
observed in X-ray crystal structures of native Drosophila GMII (dGMII). However,
a mutant enzyme in which the catalytic nucleophilic aspartate was changed to
alanine (D204A) allowed visualization of soaked oligosaccharides and led to the
identification of the binding site for the alpha(1,3)-linked mannoside of the
natural substrate. These studies also indicate that the conformational change of
the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric
hindrance from, and the formation of strong hydrogen bonds to, Asp204. The
observation that 1-thio-linked mannosides are not well tolerated by the
catalytic site of dGMII led to the synthesis of a pentasaccharide containing the
alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc
moiety proposed to be accommodated by the extended binding site of the enzyme. A
cocrystal structure of this compound with the D204A enzyme revealed the
molecular interactions with the beta(1,2)-linked GlcNAc. The structure is
consistent with the approximately 80-fold preference of dGMII for the cleavage
of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the
lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic
analysis indicates oligomannoside substrates without non-reducing-terminal
GlcNAc modifications are preferred, suggesting that selective inhibitors for
GMII could exploit the additional binding specificity of the GlcNAc binding site.
|
|
|
|
|
|
|
