Human UDP-galactose 4-Epimerase
ACCOMMODATION OF UDP-N-ACETYLGLUCOSAMINE WITHIN THE
ACTIVE SITE*
James B.
Thoden
,
Travis M.
Wohlers§¶,
Judith L.
Fridovich-Keil
, and
Hazel M.
Holden
**
From the
Department of Biochemistry, University of
Wisconsin, Madison, Wisconsin 53705 and the § Graduate
Program in Genetics and Molecular Biology and the
Department of
Genetics, Emory University School of Medicine,
Atlanta, Georgia 30322
Received for publication, January 10, 2001
 |
ABSTRACT |
UDP-galactose 4-epimerase catalyzes the
interconversion of UDP-galactose and UDP-glucose during normal
galactose metabolism. One of the key structural features in the
proposed reaction mechanism for the enzyme is the rotation of a
4'-ketopyranose intermediate within the active site pocket. Recently,
the three-dimensional structure of the human enzyme with bound NADH and
UDP-glucose was determined. Unlike that observed for the protein
isolated from Escherichia coli, the human enzyme can also
turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we
describe the three-dimensional structure of human epimerase complexed
with NADH and UDP-GlcNAc. To accommodate the additional
N-acetyl group at the C2 position of the sugar, the side
chain of Asn-207 rotates toward the interior of the protein and
interacts with Glu-199. Strikingly, in the human enzyme, the structural
equivalent of Tyr-299 in the E. coli protein is replaced
with a cysteine residue (Cys-307) and the active site volume for the
human protein is calculated to be ~15% larger than that observed for
the bacterial epimerase. This combination of a larger active site
cavity and amino acid residue replacement most likely accounts for the
inability of the E. coli enzyme to interconvert UDP-GlcNAc
and UDP-GalNAc.
 |
INTRODUCTION |
Enzymes belonging to the short chain
dehydrogenase/reductase superfamily are widespread in nature and have
been isolated from various sources including mammals, insects, and
bacteria. Members of this superfamily catalyze a wide range of
biochemical reactions with some displaying dehydrogenase activities,
and others acting as dehydratases, isomerases, or epimerases, for
example (1-3). These enzymes are typically around 250 amino acid
residues in length and contain two characteristic signature sequences.
The first of these is a YXXXK motif in which the conserved
tyrosine plays a key role in catalysis. The second of the signature
sequences is a GXXXGXG motif, which is located
near the cofactor-binding pocket.
UDP-galactose 4-epimerase, the focus of this investigation and
hereafter referred to as epimerase, is a member of the short chain
dehydrogenase/reductase superfamily. As outlined in Scheme 1, the enzyme plays a pivotal role in the
conversion of galactose to glucose 1-phosphate via the Leloir pathway.
The specific function of epimerase in this metabolic pathway is to
convert UDP-galactose back to UDP-glucose (step 3). Key features
thought to be involved in the reaction mechanism of epimerase are
indicated in Scheme 2 and include: 1)
abstraction of the 4'-hydroxyl hydrogen of the sugar, most likely by
the conserved tyrosine residue contained in the YXXXK motif
(4), 2) transfer of a hydride from C4 of the sugar to C4 of
NAD+ leading to a 4'-ketopyranose intermediate and NADH,
and finally 3) rotation of the resulting 4'-ketopyranose moiety in the
active site, thereby allowing return of the hydride from NADH to the opposite face of the sugar. Extensive x-ray crystallographic analyses with the enzyme obtained from Escherichia coli have
supported this catalytic mechanism (5-9).
Recently, the structure of human epimerase was determined and refined
to 1.5-Å resolution by x-ray diffraction (4). The human protein is a
homodimer with each subunit containing 348 amino acid residues. One
subunit of the "abortive complex" of the enzyme containing bound
NADH and UDP-glucose is shown in Fig. 1.
As can be seen, the polypeptide chain folds into two distinct regions:
the N-terminal domain (Met-1 to Thr-189) responsible primarily for
NAD+/NADH positioning and the C-terminal motif (Gly-190 to
Ala-348) involved in UDP-sugar binding. As expected for an enzyme that requires NAD+, the N-terminal domain is characterized by a
modified Rossmann fold of seven strands of parallel
-sheet flanked
on either side by
-helices. Additionally, the characteristic
YXXXK motif of the short chain dehydrogenase/reductase
superfamily is located in this domain (Tyr-157-Gly-Lys-Ser-Lys-161).
The C-terminal portion of the epimerase subunit is composed of six
-strands and five
-helices.

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Fig. 1.
Ribbon representation of one
subunit of human UDP-galactose 4-epimerase. This figure and
Figs. 3-6 were prepared with the program MOLSCRIPT (27). X-ray
coordinates utilized for this figure were obtained from the Protein
Data Bank (1EK6). Bound UDP-glucose and NADH are displayed in
ball-and-stick representations. The N-terminal domain,
delineated by Met-1 to Thr-189, is shown in blue, and the
C-terminal motif, formed by Gly-190 to Ala-348, is displayed in
green. Note that the seventh -strand in the Rossmann fold
is contributed by the C-terminal domain.
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|
Impairment of human epimerase results in epimerase deficiency
galactosemia, a variant form of galactosemia with clinical severity that ranges from apparently benign to potentially lethal (10-13). Epimerase deficiency galactosemia can affect as many as 1/6700 individuals, at least in some ethnic groups (10, 14-15). In addition to catalyzing the interconversion of UDP-galactose and UDP-glucose, the
human enzyme is also capable of interconverting UDP-GalNAc and
UDP-GlcNAc (16-20). Strikingly, this activity has not been reported
for the E. coli enzyme. To address the manner in which the
human epimerase is able to bind and interconvert UDP-GalNAc and
UDP-GlcNAc, the structure of the abortive complex of the protein with
bound NADH and UDP-GlcNAc has been solved to 1.5-Å resolution by x-ray
crystallographic methods. Here we describe the structure of this
ternary complex and compare it to that of the bacterial enzyme.
 |
EXPERIMENTAL PROCEDURES |
Crystallization of the Epimerase/UDP-GlcNAc/NADH Abortive
Complex--
Protein employed for this investigation was purified as
described previously (4). The ternary complex was prepared by treating the epimerase (15 mg/ml in the final dialysis buffer) with 5 mM NADH and 20 mM UDP-GlcNAc and allowing the
solution to stand for 24 h at 4 °C. Large crystals were grown
from 100 mM MES1
(pH 6.0), 8-9% (w/v) poly(ethylene glycol) 3400, and 75 mM MgCl2 by macroseeding into batch experiments
at 4 °C. Typically, the crystals achieved maximum dimensions of 0.1 mm × 0.1 mm × 1.0 mm in ~2-3 weeks.
X-ray Structural Analysis of the Epimerase/UDP-GlcNAc/NADH
Abortive Complex--
For x-ray data collection, the crystals were
first transferred to a synthetic mother liquor containing 15%
poly(ethylene glycol) 3400, 75 mM MgCl2, 250 mM NaCl, 5 mM NADH, and 20 mM
UDP-GlcNAc buffered at pH 6.0 with 100 mM MES. The crystals
were then transferred to a cryoprotectant solution in two steps: first
to an intermediate solution containing 20% poly(ethylene glycol) 3400, 500 mM NaCl, and 20% (v/v) methanol and then to a similar
solution that had been augmented with 4% (v/v) ethylene glycol. These
crystals were suspended in a loop of 20-µm surgical thread and
immediately flash-frozen in a stream of nitrogen gas.
The crystals belonged to the space group
P212121 with unit cell dimensions
of a = 63.4 Å, b = 88.9 Å, and
c = 118.7 Å. The asymmetric unit contained one dimer.
A native x-ray data set to 1.5-Å resolution was collected at the
Advanced Photon Source (Structural Biology Center beamline 19-BM).
These data were processed with HKL2000 and scaled with SCALEPACK (21).
Relevant x-ray data collection statistics are presented in Table
I. The structure was solved via AMORE
(22) using the previously determined epimerase/UDP-glucose/NADH structure as the starting model (4). Initial refinement with the
package TNT (23) reduced the R-factor to ~29.5% at 1.5-Å resolution. Manual adjustment of the model with the program Turbo (24)
and subsequent refinement reduced the R-factor to 18.5% for
all measured x-ray data from 30 to 1.5 Å. Relevant refinement statistics can be found in Table II.
Electron density corresponding to the UDP-GlcNAc moiety in subunit II
of the asymmetric unit is shown in Fig.
2.

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Fig. 2.
Difference electron density corresponding to
the UDP-GlcNAc moiety in subunit II of the asymmetric unit. The
map was contoured at 3 and calculated with coefficients of the form
(Fo Fc), where
Fo was the native structure factor amplitude and
Fc was the calculated structure factor
amplitude. The UDP-GlcNAc ligand was omitted from the x-ray coordinate
file for the electron density map calculation. This figure was produced
by the software package BobScript (28).
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 |
RESULTS AND DISCUSSION |
Molecular Structure of the Human Epimerase/UDP-GlcNAc/NADH
Complex--
A close-up view of the human epimerase active site with
bound UDP-GlcNAc (subunit II) is shown in Fig.
3a. Nineteen water molecules
are located within 4.0 Å of the NADH and UDP-GlcNAc moieties. Human
epimerase is known to be a B-side-specific enzyme. As
expected, the nicotinamide ring of the NADH is in the
syn-conformation with its si-face oriented toward
the sugar ligand. Specifically, the distance between C4 of the
UDP-GlcNAc ligand and C4 of the nicotinamide ring of the NADH is 3.0 Å. This distance in the previously determined human
epimerase/UDP-glucose/NADH complex is somewhat longer at 3.5 Å (4). As
indicated by the dashed lines, the 4'-hydroxyl
group of the sugar is located at 2.8 Å from O
of
Ser-132 and 3.0 Å from O
of Tyr-157. In the
epimerase/UDP-glucose/NADH model, the distance between the 4'-hydroxyl
group of the sugar and O
of Ser-132 is somewhat shorter
at ~2.4 Å while the distance between the sugar hydroxyl group and
O
of Tyr-157 is comparable at 3.1 Å (4). This close
distance of O
of Tyr-157 to the 4'-hydroxyl group of the
sugar is suggestive for the role of Tyr-157 as the catalytic base that
abstracts the hydrogen during normal catalysis.

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Fig. 3.
The active site for human epimerase with
bound NADH and UDP-GlcNAc. Those amino acid residues and solvents
located within ~4 Å of the ligands are shown in a. Both
NADH and UDP-GlcNAc are highlighted in yellow bonds.
Hydrogen bonds between the 4'-hydroxyl group of the sugar and the side
chains of Ser-132 and Tyr-157 are indicated by the dashed
lines. A schematic of the hydrogen bonding pattern around
the sugar substrate is given in b. The dashed
lines indicate distances equal to or less than 3.2 Å. The
side chain oxygens of Ser-132 and Tyr-157 are separated by 4.0 Å, as
indicated by the dashed line.
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|
A schematic of the hydrogen bonding pattern between the protein and the
sugar substrate is displayed in Fig. 3b. Eight water molecules lie within hydrogen bonding distance to the ligand. Key amino
acid side chains responsible for positioning the sugar within the
active site include Ser-132, Tyr-157, Asn-187, Arg-239, Arg-300, and
Asp-303. Additionally, the backbone carbonyl or peptidic NH groups of
Lys-92, Leu-208, Asn-224, and Phe-226 form hydrogen bonds with the
UDP-GlcNAc moiety.
The epimerase/UDP-GlcNAc/NADH abortive complex crystallized with two
molecules in the asymmetric unit. Interestingly, the
-carbons for
the two subunits superimpose with quite a large root mean square
deviation of 1.1 Å. The major differences between the two polypeptide
chains, however, are confined to the N and C termini and to the surface
loop delineated by Gly-41 to Gly-45. Shown in Fig.
4 is a close-up view of the superposition
of the two active sites contained within the asymmetric unit. Quite
unexpectedly, the sugar moieties of the two ligands adopt substantially
different orientations. In subunit I, displayed in red, C4
of the UDP-GlcNAc group is positioned at 4.6 Å from C4 of the
nicotinamide ring of the dinucleotide. Additionally, its 4'-hydroxyl
group lies at 3.3 Å from O
of Ser 132 and 4.6 Å from
O
of Tyr-157. Due to differences in the torsional angles
about the phosphate backbones of the UDP-sugars, the GlcNAc group in subunit I swings out more toward the solvent and the
N-acetyl group attached to C2 of the sugar adopts two
different conformations as depicted in Fig. 4. Clearly these observed
differences in sugar binding are in agreement with a catalytic
mechanism that requires free rotation of a 4'-ketopyranose intermediate
within the active site of the enzyme.

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Fig. 4.
Superposition of the two active sites for the
human epimerase dimer contained within the asymmetric unit.
Subunit I in the x-ray coordinate file is depicted in red,
and subunit II is displayed in black. The
asterisk marks the position of C4 in the sugar moiety bound
to subunit I.
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Comparison of the Human Epimerase/UDP-Glucose/NADH and
Epimerase/UDP-GlcNAc/NADH Complexes--
One of the questions to be
addressed by this study is the manner in which the human epimerase can
accommodate both UDP-glucose and UDP-GlcNAc. A superposition of the two
structures, namely the epimerase/UDP-glucose/NADH (subunit II) and the
epimerase/UDP-GlcNAc/NADH (subunit II) abortive complexes, is displayed
in Fig. 5. The
-carbons for these two
models superimpose with a root mean square deviation of 0.50 Å.
Clearly the more bulky N-acetyl group attached to C2 of the
sugar is accommodated within the epimerase active site by a simple
rotation of the carboxamide side chain group of Asn 207.

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Fig. 5.
Superposition of the active sites for the
abortive complexes of human epimerase with either UDP-glucose or
UDP-GlcNAc bound. The abortive complexes with bound UDP-glucose or
UDP-GlcNAc are color-coded in red and black,
respectively. Note the movement of the side chain carboxamide group of
Asn-207.
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Comparison of the Human Epimerase/UDP-GlcNAc/NADH, the E. coli
Epimerase/UDP-Glucose/NADH, and the E. coli
Epimerase/UDP-Galactose/NADH Complexes--
By far the most extensive
structural studies on UDP-galactose 4-epimerase to date have been
conducted on the enzyme from E. coli. In one study, the
structure of the bacterial enzyme/NADH/UDP-glucose abortive complex was
solved in order to address the manner in which a sugar ligand binds
within the active site (7). Interestingly, all attempts to produce a
similar abortive complex of the E. coli enzyme in the
presence of UDP-galactose failed. Although crystals could be grown of
the putative enzyme/UDP-galactose/NADH species, after x-ray data
collection and processing, UDP-glucose rather than UDP-galactose was
always observed binding in the active site. It was eventually possible
to study the manner in which UDP-galactose binds to the bacterial
enzyme, however, by preparing a "double" site-directed mutant
protein in which Ser-124 and Tyr-149 were changed to alanine and
phenylalanine residues, respectively. The x-ray crystallographic
analysis of this "double" site-directed mutant protein demonstrated
the manner in which UDP-galactose binds in the active site cleft (9).
From these analyses, it was shown that in the E. coli
epimerase, Asn-179 (which is the structural equivalent to Asn-187 in
the human enzyme) serves to hydrogen bond to either the 6'- or the
2'-hydroxyl groups when UDP-glucose or UDP-galactose, respectively, are
bound in the active site. Additionally, in the E. coli
enzyme, Tyr-299 functions in sugar positioning where it also hydrogen
bonds to either the 6'- or 2'-hydroxyl groups of UDP-glucose or
UDP-galactose, respectively. It is particularly noteworthy that Tyr-299
is not conserved in the human enzyme but rather is replaced with a
cysteine residue (Cys-307).
A superposition of the E. coli epimerase with bound
UDP-glucose onto the human epimerase with bound UDP-GlcNAc is shown in Fig. 6. The
-carbons for these two
complexes correspond with a root mean square deviation of 1.2 Å. Note
that the sugar moieties of the UDP-glucose bound to the E. coli enzyme (highlighted in blue) and the UDP-GlcNAc
bound to the human epimerase (outlined in black) adopt
similar orientations. Also shown in Fig. 6 is the conformation of
UDP-galactose when it is positioned in the active site of the E. coli enzyme (displayed in red). From Fig. 6 it is clear
that if UDP-GalNAc binds to the human enzyme in a similar manner to
that observed for UDP-galactose in the bacterial protein, the
N-acetyl group of the sugar ligand will lie quite close to
the sulfhydryl group of Cys-307. Attempts to grow crystals of an
abortive complex of the human enzyme with NADH and UDP-GalNAc have thus
far been unsuccessful. Preparation of the "double" site-directed mutant protein of the human enzyme is presently in progress whereby Ser-132 and Tyr-157 are being changed, respectively, to alanine and
phenylalanine residues. An x-ray analysis of this double site-directed mutant protein should clarify the manner in which UDP-GalNAc binds within the active site of the human enzyme.

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Fig. 6.
Superposition of human
epimerase/UDP-GlcNAc/NADH structure onto the abortive complex models of
the E. coli epimerase with bound UDP-glucose or
UDP-galactose. The human protein is shown in black, and
the E. coli protein with bound UDP-glucose is depicted in
blue and with bound UDP-galactose highlighted in
red. The numbering corresponds to the human
epimerase. Note especially the replacement of Cys-307 in the human
enzyme by a tyrosine residue (Tyr-299) in the bacterial protein.
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What is absolutely clear from this investigation is why the human
enzyme can interconvert UDP-GlcNAc and UDP-GalNAc while the bacterial
enzyme has not been reported to display such activity. Calculations
with the program VOIDOO (25, 26) indicate that the active site for the
human enzyme, as compared with that of the bacterial protein, is
~15% larger in the region responsible for sugar binding.
Furthermore, the replacement of Cys-307 in the human enzyme with the
more bulky Tyr-299 in the bacterial protein most likely precludes
UDP-GalNAc from binding in the E. coli active site in a
productive mode. By the judicious use of various site-directed mutant
proteins, it should be possible to test this hypothesis. Indeed,
experiments are presently under way to construct a form of the E. coli epimerase that is catalytically active toward UDP-GlcNAc and
UDP-GalNAc.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the helpful
discussions of Dr. W. W. Cleland. Use of the Argonne National
Laboratory Structural Biology Center beamlines at the Advanced Photon
Source was supported by the United States Department of Energy, Office
of Energy Research, under Contract W-31-109-ENG-38. We are grateful to
Drs. Dale Edmondson and Paige Newton-Vinson for generously allowing
and helping us to use their fermenter.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants DK47814 (to H. M. H.) and DK46403
(to J. L. F.-K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1HZJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶
Supported in part by funds provided by National Institutes of
Health Predoctoral Training Grant GM08490.
**
To whom correspondence should be addressed. Tel.:
608-262-4988; Fax: 608-262-1319; E-mail:
hazel_holden@biochem.wisc.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M100220200
 |
ABBREVIATIONS |
The abbreviation used is:
MES, 2-(N-morpholino)ethanesulfonic acid.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.