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PDBsum entry 1v84
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural basis for acceptor substrate recognition of a human glucuronyltransferase, Glcat-P, An enzyme critical in the biosynthesis of the carbohydrate epitope hnk-1.
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Authors
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S.Kakuda,
T.Shiba,
M.Ishiguro,
H.Tagawa,
S.Oka,
Y.Kajihara,
T.Kawasaki,
S.Wakatsuki,
R.Kato.
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Ref.
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J Biol Chem, 2004,
279,
22693-22703.
[DOI no: ]
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PubMed id
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Abstract
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The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules.
Its structure is characterized by a terminal sulfated glucuronyl acid. The
glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of
the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified
the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic
activity showed that it catalyzed the transfer reaction for N-acetyllactosamine
(Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor
substrate. Subsequently, we determined the first x-ray crystal structures of
human GlcAT-P, in the absence and presence of a donor substrate product UDP,
catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine
(Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The
asymmetric unit contains two independent molecules. Each molecule is an
alpha/beta protein with two regions that constitute the donor and acceptor
substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized
by conserved amino acid residues including a DXD motif
(Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with
the terminal galactose moiety of the acceptor substrate. In addition, Val(320)
and Asn(321), which are located on the C-terminal long loop from a neighboring
molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These
three residues play a key role in establishing the acceptor substrate
specificity.
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Figure 3.
FIG. 3. The electron density maps of the substrates and
cofactor. A, the omit F[O]-F[C] electron density map of the UDP
molecule and Mn2+ ion, contoured at 1.6  (gray) and 6.0 (blue),
respectively, superimposed with a ball-and-stick model colored
according to atom types (nitrogen, blue; carbon, black; oxygen,
red; phosphorous, purple; manganese, orange). B, the omit
F[O]-F[C] electron density map of the N-acetyllactosamine,
contoured at 1.6 (gray), superimposed
with a ball-and-stick model. C, the interactions between Mn2+,
UDP, and Asp197 side chain of GlcAT-P. The Mn2+ interactions are
shown in blue dashed lines.
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Figure 4.
FIG. 4. Comparison of GlcAT-P quaternary complex (UDP,
Mn2+, and N-acetyllactosamine) with GlcAT-I quaternary complex
(UDP, Mn2+, and Gal  1-3Gal). A, dimer
structure of GlcAT-P complex. Each monomer is colored blue and
yellow, respectively. Substrate molecules are shown in
ball-and-stick models. B, dimer structure of GlcAT-I complex is
shown in the same orientation as in A. C, dimer surface of
GlcAT-P complex is colored according to the electrostatic
surface potential (blue, positive; red, negative; scale from -10
to +10 kT/e). D, surface representation of GlcAT-I complex in
dimer is shown in the same orientation as in C.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
22693-22703)
copyright 2004.
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