From the Department of Biochemistry, University of
Wisconsin, Madison, Wisconsin, 53706 and the § Graduate
Program in Genetics and Molecular Biology and
Department of
Genetics, Emory University School of Medicine,
Atlanta, Georgia 30322
Received for publication, February 9, 2001
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ABSTRACT |
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Galactosemia is an inherited disorder
characterized by an inability to metabolize galactose. Although
classical galactosemia results from impairment of the second enzyme of
the Leloir pathway, namely galactose-1-phosphate uridylyltransferase,
alternate forms of the disorder can occur due to either galactokinase
or UDP-galactose 4-epimerase deficiencies. One of the more severe cases
of epimerase deficiency galactosemia arises from an amino acid
substitution at position 94. It has been previously demonstrated that
the V94M protein is impaired relative to the wild-type enzyme
predominantly at the level of Vmax rather than
Km. To address the molecular consequences the
mutation imparts on the three-dimensional architecture of the enzyme,
we have solved the structures of the V94M-substituted human
epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val94 packs near the aromatic group of the
catalytic Tyr157 and serves as a molecular "fence" to
limit the rotation of the glycosyl portions of the UDP-sugar substrates
within the active site. The net effect of the V94M substitution is an
opening up of the Ala93 to Glu96 surface loop,
which allows free rotation of the sugars into nonproductive binding modes.
Galactosemia is a rare, potentially lethal genetic disease that is
inherited as an autosomal recessive trait and results in the inability
of patients to properly metabolize galactose (1). Clinical
manifestations include intellectual retardation, liver dysfunction, and
cataract formation, among others. Although deficiencies of any of the
three enzymes participating in the Leloir pathway for galactose
metabolism (Scheme 1) can result in
symptoms of galactosemia, the classical form of the disease arises from
impairment of galactose-1-phosphate uridylyltransferase, the second
enzyme in the pathway (1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Scheme 1.
Of particular interest is the third enzyme in the pathway, namely UDP-galactose 4-epimerase, hereafter referred to as epimerase. This NAD+-dependent enzyme plays a key role in normal galactose metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose as indicated in Scheme 1. Interestingly, the human form of epimerase has also been shown to interconvert UDP-GlcNAc and UDP-GalNAc (2-4). This type of activity has not been observed in the epimerase from Escherichia coli.
Two types of human epimerase-based galactosemia have been identified thus far: peripheral and generalized. While the peripheral form can be quite common among some ethnic groups and is usually considered benign, the generalized form of the disease is clinically severe and extremely rare (5-8). The most severe form of epimerase deficiency galactosemia characterized to date arises from a homozygous mutation encoding the substitution of a methionine residue for a valine at position 94 (9). This substitution impairs enzyme activity to ~5% of wild-type levels with respect to UDP-galactose and to ~25% of wild-type levels with respect to UDP-GalNAc (10). The mutant protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather than Km (10).
Previous biochemical analyses on the epimerase from E. coli
have suggested that its reaction mechanism proceeds through abstraction of the hydrogen from the 4'-hydroxyl group of the sugar by a catalytic base and transfer of a hydride from C-4 of the sugar to C-4 of the NAD+, leading to a 4'-ketopyranose intermediate and
NADH (11). A limited but well-defined rotation of this intermediate is
thought to occur in the active site, thereby allowing return of the
hydride from NADH to the opposite side of the sugar. Recently, the
three-dimensional structure of human epimerase complexed with NADH and
UDP-glucose was solved by x-ray crystallographic analyses to 1.5-Å
resolution (12). A ribbon representation of one subunit of the
homodimeric protein is displayed in Fig. 1. As can be seen, the overall
fold of the enzyme can be described in terms of two structural motifs: the N-terminal domain defined by Met1-Thr189
and the C-terminal region formed by
Gly190-Ala348. The N-terminal domain adopts
the three-dimensional architecture referred to as a Rossmann fold.
Strikingly, the NADH and UDP-glucose ligands are positioned within the
active site such that C-4 of the sugar lies at ~3.5 Å from C-4 of
the dinucleotide (12). Additionally, O of
Tyr157 and O
of Ser132 are
located at 3.1 Å and 2.4 Å, respectively, from the 4'-hydroxyl group
of the sugar moiety. It is believed that the low barrier hydrogen bond
formed between the sugar and the side chain of Ser132
facilitates the removal of the 4'-hydroxyl hydrogen by the phenolic acid chain of Tyr157 and the transfer of the hydride from
C-4 of the sugar to C-4 of the nicotinamide ring (12).
To address the molecular consequences of the V94M substitution in human
epimerase, we have crystallized and solved the x-ray structures of the
mutant protein complexed with UDP-glucose, UDP-galactose, UDP-GlcNAc,
or UDP-GalNAc, all to 1.5-Å resolution. These investigations have
allowed for a more complete understanding of the three-dimensional consequences this mutation imparts on the active site geometry of the
enzyme and provide a molecular explanation for the observed enzymatic impairment.
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EXPERIMENTAL PROCEDURES |
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Crystallization of the Epimerase (V94M Mutant)·NADH·UDP-sugar Ternary Complexes-- The V94M form of human UDP-galactose 4-epimerase was constructed and overexpressed in the yeast Pichia pastoris, as described (9, 12). Protein samples employed for crystallization trials were purified according to the protocol of Ref. 12. Ternary complexes of the protein were prepared by treating the epimerase samples (15 mg/ml in the final dialysis buffer) with 5 mM NADH and 20 mM UDP-sugars and allowing the solutions to equilibrate for 24 h at 4 °C. Large crystals of each of the complexes 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 grew to maximum dimensions of 0.3 × 0.2 × 0.7 mm in ~1-2 weeks.
X-ray Structural Analyses of the Ternary Complexes-- For x-ray data collection, the crystals were transferred to cryoprotectant solutions in two steps. First they were slowly transferred to intermediate solutions containing 20% poly(ethylene glycol) 3400, 500 mM NaCl, and 20% (v/v) methanol. After equilibration in the methanol-containing solutions, the crystals were subsequently transferred into similar solutions that had been augmented with 4% (v/v) ethylene glycol. All of the crystals were suspended in loops of 20-µm surgical thread and immediately flash-frozen in a stream of nitrogen gas.
The four V94M protein·NADH·UDP-sugar complexes crystallized in the space group P212121 with typical unit cell dimensions of a = 78.1 Å, b = 89.9 Å, and c = 96.9 Å. Each asymmetric unit contained one dimer, and only subunit I in the coordinate file will be discussed under "Results and Discussion." Native x-ray data sets to 1.5-Å resolution were collected at the Advanced Photon Source, Structural Biology Center beamline 19-BM. These data were processed with HKL2000 and scaled with SCALEPACK (13). Relevant x-ray data collection statistics are presented in Table I. The V94M protein·NADH·UDP-glucose structure was solved via AMORE (14), employing the previously determined wild-type protein·NADH·UDP-glucose structure as the search model (12). The other three structures were solved via difference Fourier techniques. Manual adjustments of the models using the program Turbo (15) and subsequent least squares refinements with the package TNT (16) reduced the R-factors to 17.5, 18.1, 18.5, and 17.8%, respectively, for the ternary complexes with UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. Relevant refinement statistics can be found in Table II. Figs. 1, 2, 3, and 5 were prepared with the software package, MOLSCRIPT while Fig. 4 was generated with the program BobScript (17, 18).
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RESULTS AND DISCUSSION |
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Recent biochemical studies on the human V94M-substituted UDP-galactose 4-epimerase revealed that the enzyme was kinetically impaired relative to the wild-type protein predominantly at the level of Vmax rather than Km (10). Specifically, the Km values for the wild-type and mutant forms of the protein, when assayed with UDP-galactose, were 0.27 ± 0.01 and 0.15 ± 0.02 mM, respectively. The Vmax values, however, were significantly different for the wild-type and V94M proteins at 1.22 versus 0.036 mmol of UDP-galactose/mg/min, respectively. Additionally, the apparent Km values for the wild-type and V94M enzymes, with UDP-GalNAc as the substrate, corresponded to 0.287 ± 0.05 mM and 0.445 ± 0.01 mM, respectively.
For the x-ray investigation presented here, four different crystal
forms of the V94M enzyme were prepared, namely the complexes of protein
with NADH and the following ligands: UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-galNAc. All of the structures were solved to 1.5-Å
resolution and refined to R-factors equal to or less than 18.5%. The relative location of the galactosemic mutation with respect
to the active site of the native enzyme can be seen in Fig.
2a. As expected, the main
structural perturbation imposed by the V94M substitution occurs in the
helical loop defined by Ala93 to Glu96, which
connects the fourth -strand to the fourth major
-helix of the
Rossmann fold. This change in loop structure is similar in all of the
V94M protein models described here and is independent of the identity
of the sugar ligand occupying the active site. In the wild-type enzyme,
the side chain of Val94 points toward the active site and
is located at ~3.5 Å from the catalytic Tyr157.
Additionally, the carbonyl oxygen of Val94 forms a hydrogen
bond with the side chain hydroxyl group of Ser97, which
further serves to tighten down that portion of the polypeptide chain
backbone abutting the UDP-sugar binding pocket. This loop in the
wild-type enzyme is well ordered with an average temperature factor of
22.0 Å2 for all of the atoms lying between
Ala93 and Glu96. The corresponding temperature
factors for the V94M protein·NADH·UDP-sugar complexes are
significantly higher, however, at ~77 Å2. Indeed,
residual electron density in maps calculated with
(Fo
Fc) coefficients suggests
that alternate conformations of this loop are present in the
crystalline lattice but at lower occupancies. It was not possible to
build these alternate conformations into the electron density with any
certainty, however, and hence they were not included in the protein
models.
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A superposition of the polypeptide chains near residue 94 for the
wild-type enzyme and the V94M protein complexed with UDP-galactose is
displayed in Fig. 2b. The mutation at position 94 results in a significant change in the backbone dihedral angles of the preceding alanine residue. Specifically, in the wild-type enzyme,
Ala93 adopts and
angles of approximately
82 and
106°, respectively, while in the V94M protein, the corresponding
angles are
124° and 148°. As a result of these changes in
torsional angles, the side chain of Met94 in the mutant
protein extends out toward the solvent, and the hydrogen bond between
the carbonyl oxygen of residue 94 and O
of
Ser97 is no longer present. It should be noted that if the
loop between Ala93 and Glu96 were to adopt the
wild-type conformation in the V94M protein, the larger side chain of
Met94 could not be accommodated in the active site without
significant steric clashes, and this, presumably, explains in
part the dramatic changes in backbone conformation starting at
position 93.
The net effect of this three-dimensional perturbation is an opening of
the active site, thereby allowing free rotation of the sugar moiety.
Indeed, from electron density maps calculated with
(Fo Fc) coefficients, it is
clear that the sugars in the V94M structures complexed with either
UDP-glucose or UDP-galactose adopt multiple conformations that are not
well defined. Because of this, it is not possible to describe in detail the carbohydrate/protein interactions in these two particular structures. It is possible, however, to compare the interactions between the protein and the UDP moieties in the wild-type and the V94M
proteins with either bound UDP-glucose or UDP-galactose. Potential
hydrogen-bonding interactions observed between the wild-type enzyme and
the substrate are depicted in a schematic representation in Fig.
3a, while those for the
V94M·NADH·UDP-glucose complex are shown in Fig. 3b. As
indicated, the side chains forming hydrogen bonds to the UDP-glucose in
the wild-type protein include Ser132, Tyr157,
Asn187, Asn207, Arg239,
Asp303, and Arg300. Only Ser132 and
Tyr157 interact solely with the carbohydrate portion. All
of the other side chains are primarily involved in UDP binding. As can
be seen in Fig. 3a, the uracil ring of the UDP-glucose is
anchored to the native enzyme via the backbone carbonyl group of
Asn224 and the peptidic NH group of Phe226. Two
additional water molecules serve to bridge the C-4 carbonyl group of
the base to the protein. These interactions are also observed in the
various V94M mutant protein models (Fig. 3b). In the
wild-type protein, the 2'- and 3'-hydroxyl groups of the uridine ribose
are hydrogen-bonded to the carboxylate group of Asp303 and
a water molecule, respectively. The guanidinium group of Arg300 interacts with both
- and
-phosphoryl oxygens
of UDP when bound to wild-type enzyme, while the side chain of
Arg239 forms an electrostatic interaction with a
-phosphoryl oxygen. Similar interactions are, indeed, observed in
the V94M enzymes as indicated in Fig. 3b. The only
significant differences between the wild-type enzyme and the V94M
protein occur at the glucose moiety. In the wild-type protein, the
glucose moiety is firmly anchored in place by interactions with the
side chains of Ser132 and Tyr157 and the
carbonyl oxygen of Lys92, which is located in the loop
containing the V94M mutation. These interactions are missing in the
mutant proteins with bound UDP-glucose or UDP-galactose due to the free
rotation of the glycosyl groups in the active site. The limited change
in Km observed between the wild-type enzyme and the
V94M protein is a function of the fact that the nucleotide portion of
the UDP-sugar substrate provides most of the binding interactions.
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Unlike that observed for the V94M protein·NADH·UDP-glucose or the
V94M protein·NADH·UDP-galactose complexes, the sugar moieties in
the V94M proteins with either bound UDP-GlcNAc or UDP-GalNAc are
visible in the electron density maps as shown in Fig.
4 for the UDP-GlcNAc species. Note that
the 6'-hydroxyl group of the N-acetylglucosamine adopts two
conformations and that the sugar is rotated away from the nicotinamide
ring of the NADH. Residual electron density in maps calculated with
(Fo Fc) coefficients suggests
that each of these sugars adopt alternate conformations at lower
occupancies. Because of the quality of the residual electron density,
however, it was not possible to unambiguously model these alternate
conformations into the electron density; hence, they were not included
in the coordinate files.
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Interestingly, the electron density map calculated for the V94M enzyme crystallized in the presence of UDP-GalNAc clearly demonstrated that the ligand had been converted to UDP-GlcNAc. This result is reminiscent of that observed with the epimerase from E. coli. All attempts to prepare an abortive complex of the bacterial enzyme with UDP-galactose failed (19). These experiments included reduction of the enzyme with dimethylamine/borane in the presence of UDP-galactose, UDP, UMP, or TMP and subsequent exchange of these nucleotides with UDP-galactose. In every case, the electron density maps always indicated the presence of UDP-glucose in the active site. Obviously, UDP-glucose binds more tightly to epimerase in the abortive complex, and although the enzyme had been reduced with dimethylamine/borane, enough residual activity remained to convert UDP-galactose to UDP-glucose. Most likely, the same phenomenon is occurring in the case of the human V94M-substituted epimerase with bound UDP-GalNAc.
Within the last year, the three-dimensional structure of human
wild-type epimerase with bound NADH and UDP-GlcNAc was solved to 1.5-Å
resolution, and it was demonstrated that to accommodate the additional
N-acetyl group at the C-2 position of the sugar, the side
chain of Asn207 rotates toward the interior of the protein
and interacts with Glu199 (20). Shown in Fig.
5 is a superposition of the active site regions for the wild-type and V94M proteins with bound UDP-GlcNAc. As
can be seen, in the V94M-substituted form, the sugar group of the
ligand rotates out of the pocket and toward position 94. This type of
rotation is blocked in the native enzyme due to the side chain of
Val94. In the wild-type enzyme the distance between C-4 of
the UDP-GlcNAc ligand and C-4 of the nicotinamide ring of the NADH is
3.0 Å. This distance in the V94M protein model is 9.4 Å. The
4'-hydroxyl group of the sugar in the wild-type enzyme is located at
2.8 Å from O of Ser132 and 3.0 Å from
O
of Tyr157. Due to the drastic rotation of
the sugar moiety in the active site pocket, these distances in the V94M
protein complex are 9.5 and 10.2 Å, respectively. Key hydrogen bonds
between the N-acetylglucosamine group of the ligand and the
wild-type enzyme occur between the side chains of Asn187
and the 6'-OH of the sugar, between both Ser132 and
Tyr157 and the 4'-OH of the sugar, and finally, between the
carbonyl group of Lys92 and the 3'-OH of the carbohydrate.
These interactions are completely missing in the V94M enzyme with bound
UDP-GlcNAc, where the hydroxyl groups simply form hydrogen bonds with
solvent molecules.
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In summary, the x-ray studies described here provide a
three-dimensional understanding of one example of severe epimerase deficiency galactosemia. In the normal enzyme, the hydrophobic side
chain of Val94 provides a "molecular fence" to prevent
sugar rotation out of the active site pocket, thereby preventing
nonproductive binding. Upon substitution of Val94 by a
methionine, the loop region connecting the fourth -strand to the
fourth
-helix of the Rossmann fold becomes disordered, adopts
multiple conformations, and effectively opens up the sugar binding
pocket to allow for free rotation of the sugar moiety in the active
site and/or nonproductive substrate binding. In light of the structural
results presented here, it is not surprising that the V94M mutation
effects Vmax significantly more than
Km. Most of the binding interactions for the
UDP-sugar substrates occur between the protein and the nucleotide, and
these are not disrupted by the mutation. What the V94M substitution
does, however, is allow the carbohydrate portions of the UDP-sugars to
rotate freely, thereby limiting the time the ligand is bound in a
productive mode near O
of Ser132 and
O
of Tyr157. As such,
Vmax is severely affected. Interestingly, in
previous work, it has been shown that the V94M epimerase is impaired to a 5-fold lesser extent with regard to UDP-GalNAc than to UDP-galactose (9). The reason is, presumably, that the bulkier sugar moieties of
UDP-GlcNAc and UDP-GalNAc can adopt fewer nonproductive binding modes.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Dale Edmondson and Paige Newton-Vinson for generously allowing and helping us to use the fermenter and to Dr. W. W. Cleland for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health (NIH) Grants DK47814 (to H. M. H.) and DK46403 (to J. L. F-K.). 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.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 1I3M, 1I3N, 1I3K, and 1I3L) 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 NIH 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, March 7, 2001, DOI 10.1074/jbc.M101304200
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ABBREVIATIONS |
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The abbreviation used is: MES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
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