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PDBsum entry 1kvq
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* Residue conservation analysis
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DOI no:
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Biochemistry
36:10685-10695
(1997)
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PubMed id:
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Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli.
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J.B.Thoden,
A.M.Gulick,
H.M.Holden.
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ABSTRACT
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UDP-galactose 4-epimerase plays a critical role in sugar metabolism by
catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it
was assumed that the enzyme contained a "traditional" catalytic base that served
to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or
UDP-galactose substrates during the course of the reaction. However, recent
high-resolution X-ray crystallographic analyses of the protein from Escherichia
coli have demonstrated the lack of an aspartate, a glutamate, or a histidine
residue properly oriented within the active site cleft for serving such a
functional role. Rather, the X-ray crystallographic investigation of the
epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that
both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'-
and 3'-hydroxyl groups of the sugar, respectively. To test the structural role
of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant
proteins, namely S124A, S124T, and S124V, were constructed and crystals of the
S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and
S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in
this investigation belonged to the space group P3221 with the following unit
cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric
unit. X-ray data sets were collected to at least 2.15 A resolution, and each
protein model was subsequently refined to an R value of lower than 19.0% for all
measured X-ray data. The investigations described here demonstrate that the
decreases in enzymatic activities observed for these mutant proteins are due to
the loss of a properly positioned hydroxyl group at position 124 and not to
major tertiary and quaternary structural perturbations. In addition, these
structures demonstrate the importance of a hydroxyl group at position 124 in
stabilizing the anti conformation of the nicotinamide ring as observed in the
previous structural analysis of the epimerase.NADH. UDP complex.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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D.J.Sukovich,
J.L.Seffernick,
J.E.Richman,
J.A.Gralnick,
and
L.P.Wackett
(2010).
Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of OleA.
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Appl Environ Microbiol,
76,
3850-3862.
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K.L.Kavanagh,
H.Jörnvall,
B.Persson,
and
U.Oppermann
(2008).
Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.
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Cell Mol Life Sci,
65,
3895-3906.
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P.Z.Gatzeva-Topalova,
A.P.May,
and
M.C.Sousa
(2005).
Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance.
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Structure,
13,
929-942.
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PDB codes:
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J.M.Schulz,
A.L.Watson,
R.Sanders,
K.L.Ross,
J.B.Thoden,
H.M.Holden,
and
J.L.Fridovich-Keil
(2004).
Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase.
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J Biol Chem,
279,
32796-32803.
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N.A.Webb,
A.M.Mulichak,
J.S.Lam,
H.L.Rocchetta,
and
R.M.Garavito
(2004).
Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway.
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Protein Sci,
13,
529-539.
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PDB code:
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N.Ishiyama,
C.Creuzenet,
J.S.Lam,
and
A.M.Berghuis
(2004).
Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa: substrate specificity in udp-hexose 4-epimerases.
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J Biol Chem,
279,
22635-22642.
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PDB codes:
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P.Z.Gatzeva-Topalova,
A.P.May,
and
M.C.Sousa
(2004).
Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain. A key enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance.
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Biochemistry,
43,
13370-13379.
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PDB code:
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H.M.Holden,
I.Rayment,
and
J.B.Thoden
(2003).
Structure and function of enzymes of the Leloir pathway for galactose metabolism.
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J Biol Chem,
278,
43885-43888.
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B.Guillot,
C.Lecomte,
A.Cousson,
C.Scherf,
and
C.Jelsch
(2001).
High-resolution neutron structure of nicotinamide adenine dinucleotide.
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Acta Crystallogr D Biol Crystallogr,
57,
981-989.
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E.Berger,
A.Arabshahi,
Y.Wei,
J.F.Schilling,
and
P.A.Frey
(2001).
Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149.
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Biochemistry,
40,
6699-6705.
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J.B.Thoden,
T.M.Wohlers,
J.L.Fridovich-Keil,
and
H.M.Holden
(2000).
Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.
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Biochemistry,
39,
5691-5701.
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PDB codes:
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M.F.Giraud,
and
J.H.Naismith
(2000).
The rhamnose pathway.
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Curr Opin Struct Biol,
10,
687-696.
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S.T.Allard,
M.F.Giraud,
C.Whitfield,
P.Messner,
and
J.H.Naismith
(2000).
The purification, crystallization and structural elucidation of dTDP-D-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-L-rhamnose synthesis pathway from Salmonella enterica serovar typhimurium.
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Acta Crystallogr D Biol Crystallogr,
56,
222-225.
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A.M.Mulichak,
M.J.Theisen,
B.Essigmann,
C.Benning,
and
R.M.Garavito
(1999).
Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose.
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Proc Natl Acad Sci U S A,
96,
13097-13102.
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PDB code:
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R.Muñoz,
R.López,
M.de Frutos,
and
E.García
(1999).
First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1.
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Mol Microbiol,
31,
703-713.
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T.M.Wohlers,
N.C.Christacos,
M.T.Harreman,
and
J.L.Fridovich-Keil
(1999).
Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.
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Am J Hum Genet,
64,
462-470.
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J.B.Thoden,
and
H.M.Holden
(1998).
Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli.
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Biochemistry,
37,
11469-11477.
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PDB codes:
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M.Rizzi,
M.Tonetti,
P.Vigevani,
L.Sturla,
A.Bisso,
A.D.Flora,
D.Bordo,
and
M.Bolognesi
(1998).
GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily.
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Structure,
6,
1453-1465.
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PDB code:
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Y.Liu,
J.B.Thoden,
J.Kim,
E.Berger,
A.M.Gulick,
F.J.Ruzicka,
H.M.Holden,
and
P.A.Frey
(1997).
Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli.
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Biochemistry,
36,
10675-10684.
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PDB code:
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
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