 |
PDBsum entry 1e7r
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Probing the catalytic mechanism of gdp-4-Keto-6-Deoxy-D-Mannose epimerase/reductase by kinetic and crystallographic characterization of site-Specific mutants.
|
|
|
Authors
|
|
C.Rosano,
A.Bisso,
G.Izzo,
M.Tonetti,
L.Sturla,
A.De flora,
M.Bolognesi.
|
|
|
Ref.
|
|
J Mol Biol, 2000,
303,
77-91.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme
responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate
of fucosyl transferases. Several cell-surface antigens, including the leukocyte
Lewis system and cell-surface antigens in pathogenic bacteria, depend on the
availability of GDP-l-fucose for their expression. Therefore, the enzyme is a
potential target for therapy in pathological states depending on
selectin-mediated cell-to-cell interactions. Previous crystallographic
investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase
belongs to the short-chain dehydrogenase/reductase protein homology family. The
enzyme active-site region is at the interface of an N-terminal NADPH-binding
domain and a C-terminal domain, held to bind the substrate. The design,
expression and functional characterization of seven site-specific mutant forms
of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In
parallel, the crystal structures of the native holoenzyme and of three mutants
(Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1.
45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 %
and 13.9 %). The refined protein models show that besides the active-site
residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic
activity and may affect the coenzyme binding mode, side-chains capable of proton
exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose)
binding pocket, are selectively required during the epimerization and reduction
steps. Among these, Cys109 and His179 may play a primary role in proton exchange
between the enzyme and the epimerization catalytic intermediates. Finally, the
additional role of mutated active-site residues involved in substrate
recognition and in enzyme stability has been analyzed.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1. The GDP- Image -fucose biosynthetic pathway and
the products obtained after NaBH[4] reduction of the
intermediate compounds. GMD, GDP- Image -mannose 4,6
dehydratase; GMER, GDP-4-keto-6-deoxy- Image -mannose
epimerase/reductase; R-, GDP.
|
|
Figure 6.
Figure 6. Stereo view of the proposed binding mode for a
nucleotide-sugar molecule (shown as a space-filling model)
relative to active-site residues discussed in the text. The C-2
and C-3 centers can be recognized as those closest to His179
side-chain; the C-4 center falls next to the nicotinamide
carboxamido group.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2000,
303,
77-91)
copyright 2000.
|
|
|
|
|
|
|
