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PDBsum entry 1vzt
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References listed in PDB file
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Key reference
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Title
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Roles of individual enzyme-Substrate interactions by alpha-1,3-Galactosyltransferase in catalysis and specificity.
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Authors
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Y.Zhang,
G.J.Swaminathan,
A.Deshpande,
E.Boix,
R.Natesh,
Z.Xie,
K.R.Acharya,
K.Brew.
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Ref.
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Biochemistry, 2003,
42,
13512-13521.
[DOI no: ]
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PubMed id
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Abstract
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The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT),
is mutationally inactivated in humans, leading to the presence of circulating
antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes
galactose transfer from UDP-Gal to beta-linked galactosides, such as lactose,
and in the absence of an acceptor substrate, to water at a lower rate. We have
used site-directed mutagenesis to investigate the roles in catalysis and
specificity of residues in alpha3GT that form H-bonds as well as other
interactions with substrates. Mutation of the conserved Glu(317) to Gln weakens
lactose binding and reduces the k(cat) for galactosyltransfer to lactose and
water by 2400 and 120, respectively. The structure is not perturbed by this
substitution, but the orientation of the bound lactose molecule is changed. The
magnitude of these changes does not support a previous proposal that Glu(317) is
the catalytic nucleophile in a double displacement mechanism and suggests it
acts in acceptor substrate binding and in stabilizing a cationic transition
state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of
this bond also linked to a conformational change in the C-terminal region of
alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280),
which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal,
is a key residue in the stringent donor substrate specificity through its role
in stabilizing the bound UDP-Gal in a suitable conformation for catalysis.
Mutation of Gln(247), which forms multiple interactions with acceptor
substrates, to Glu reduces the catalytic rate of galactose transfer to lactose
but not to water. This mutation is predicted to perturb the orientation or
environment of the bound acceptor substrate. The results highlight the
importance of H-bonds between enzyme and substrates in this glycosyltransferase,
in arranging substrates in appropriate conformations and orientation for
efficient catalysis. These factors are manifested in increases in catalytic rate
rather than substrate affinity.
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Secondary reference #1
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Title
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Structure of udp complex of udp-Galactose:beta-Galactoside-Alpha -1,3-Galactosyltransferase at 1.53-A resolution reveals a conformational change in the catalytically important c terminus.
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Authors
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E.Boix,
G.J.Swaminathan,
Y.Zhang,
R.Natesh,
K.Brew,
K.R.Acharya.
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Ref.
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J Biol Chem, 2001,
276,
48608-48614.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. a, structure of  3GT with
bound UDP and Mn2+ ion. The bound ligand and ion identify the
location of the active site. The Mn2+ ion is shown as a magenta
sphere, UDP is brown, and helices are pink, while the strands
are green. This image was created using the program MOLSCRIPT
(38). b, the amino acid sequence of the catalytic domain of 3GT with
all secondary structure elements highlighted. UDP binding
residues are marked in yellow, while the Mn2+ binding residues
are shown by closed magenta spheres. This image was created
using the program ALSCRIPT (39). c, stereoview comparison of the
C^  atoms
of form-II 3GT
(present structure, in red) with the previously determined form
I 3GT
structure (Ref. 18; in black). The C-terminal residues 358-368
in form II show a large difference in conformation and form a
lid for the active site tunnel. This image was created using the
program BOBSCRIPT (40).
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Figure 2.
Fig. 2. a, schematic figure showing the main hydrogen
bond interactions between UDP and 3GT
residues at the catalytic site of the enzyme. The Mn2+ ion and
water molecules are also shown. This image was created using the
program MOLSCRIPT (38) and rendered using Raster3D (41). b, the
location of UDP molecule in the active site tunnel. This image
was created using the program DINO (A. Philippsen; available on
the World Wide Web at www.dino3d.org).
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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Bovine alpha1,3-Galactosyltransferase catalytic domain structure and its relationship with abo histo-Blood group and glycosphingolipid glycosyltransferases.
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Authors
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L.N.Gastinel,
C.Bignon,
A.K.Misra,
O.Hindsgaul,
J.H.Shaper,
D.H.Joziasse.
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Ref.
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EMBO J, 2001,
20,
638-649.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2 Close-up stereoview of the  3GalT
UDP-Gal-binding site. (A) Hg-UDP-Gal is shown in ball-and-stick
form and color coded depending on the nature of the atoms; the
Mn2+ ion is shown as a pink sphere. Amino acid side chains
interacting with Hg-UDP-Gal are shown in ball-and-stick form in
yellow. The acidic residues from the motifs D225VD227 and the
D316E317 are shown in ball-and-stick form in red. The four amino
acid side chains of 3GalT
residues at positions equivalent to the residues distinguishing
human A-GT from B-GT are shown in ball-and-stick form in blue.
(B) Stereoview of the electron density map (2F[o] - F[c], 1  )
of the Hg-UDP-Gal-binding site.
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Figure 6.
Figure 6 Schematic representation of the 3GalT-retaining
reaction mechanism. Steps (A) and (B) are derived from the
substrate-bound 3GalT
structure. The acceptor substrate schematized in steps (C) and
(D) is a lactosamine-type glycan (Gal  1,4GlcNAc-R).
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by Macmillan Publishers Ltd
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