From the Department of Biochemistry and Molecular
Biology, University of British Columbia, 2146 Health Sciences Mall,
Vancouver V6T 1Z3, Canada and the ¶ National Research Council of
Canada, Institute of Biological Sciences, 100 Sussex Dr., Ottawa K1A
0R6, Canada
Received for publication, August 9, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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The x-ray crystallographic structure of
selenomethionyl cytosine-5'-monophosphate-acylneuraminate synthetase
(CMP-NeuAc synthetase) from Neisseria meningitidis has been
determined at 2.0-Å resolution using multiple-wavelength anomalous
dispersion phasing, and a second structure, in the presence of the
substrate analogue CDP, has been determined at 2.2-Å resolution by
molecular replacement. This work identifies the active site residues
for this class of enzyme for the first time. The detailed interactions
between the enzyme and CDP within the mononucleotide-binding pocket are
directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells
and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B
streptococci, etc.). As such, the identification of the bacterial
CMP-NeuAc synthetase active site can serve as a starting point for
rational drug design strategies.
Cytosine-5'-monophosphate-acylneuraminate synthetase
(cytosine-5'-monophosphate-N-acetyl neuraminic acid
synthetase, CMP-NeuAc synthetase)1 catalyzes the
penultimate step in the addition of sialic acids to the oligosaccharide
component of glycoconjugates and is a required component of sialylation
pathways. CMP-NeuAc synthetase-deficient mutants do not express
sialylated glycoconjugates and can be complemented with functional
CMP-NeuAc synthetase in both mammalian (1) and bacterial systems (2).
Sialic acids participate in a myriad of signaling, recognition, and
cell-cell adhesion phenomena in mammalian cells (3) and are
overexpressed in some highly malignant tumors (4). Genetic disorders
that lead to altered physiological levels of sialic acids have many
consequences in humans and can be fatal (5). In bacterial systems,
sialic acids are less common and frequently have roles as virulence
factors (6). In Neisseria gonorrhoeae, sialylated
glycoconjugates protect the organism from phagocytosis and increase
serum resistance (7). In Haemophilus ducreyi the presence of
2-keto-3-deoxy-manno-octulosonate-containing glycoconjugates correlate
with the organism's pathogenicity (8). Likewise, the sialylated
capsule of Neisseria meningitidis, Escherichia coli K1, and group B streptococci are virulence factors (9-11). It has been suggested that bacterial species produce sialylated glycoconjugates to mimic the host and escape detection by the immune
system (12). Clearly, the mechanism of production of sialic
acid-containing glycoconjugates is of clinical interest. Although
bacterial and eucaryotic CMP-NeuAc synthetase enzymes share many
catalytic properties, several important differences have been reported,
including substrate specificity, tertiary structure, inhibitor
sensitivity, and cellular localization (13). As a result, bacterial
CMP-NeuAc synthetase enzymes can be targeted by rational drug design strategies.
CMP-NeuAc synthetase is also of considerable interest in the field of
biotechnology. In the presence of Mg2+, CMP-NeuAc
synthetase utilizes CTP and acylneuraminate (NeuAc, a sialic acid) as
substrates and produces pyrophosphate and cytosine-5'-monophosphate acylneuraminate (CMP-NeuAc or CMP-NANA), an "activated" sugar nucleotide monophosphate (Fig. 1) (14,
15). The reaction catalyzed by CMP-NeuAc synthetase (and homologous
enzymes) is unique in that sialic acid is directly coupled to a
cytosine monophosphate. All other activated sugar nucleotide
synthetases use a phosphorylated sugar substrate and produce a sugar
nucleotide diphosphate (16). Given the expense of chemically
synthesizing CMP-NeuAc and its instability, CMP-NeuAc synthetase
together with various sialyltransferases are valuable for the
preparative enzymatic synthesis of biologically relevant, sialylated
oligosaccharides (17). In turn, these sialylated oligosaccharides are
used in clinical medicine and are invaluable tools for the study of the
many processes that require sialic acids.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The reaction catalyzed by CMP-NeuAc
synthetase in the presence of Mg2+. CMP-NeuAc
synthetase specifically utilizes the anomer of NeuAc (a sialic
acid) and CTP as substrates and produces pyrophosphate and CMP-NeuAc,
an activated sugar monophosphate nucleotide. In turn, CMP-NeuAc is
substrate for sialyltransferases that add sialic acids to cellular
glycoconjugates. Selected NeuAc and CTP atoms are labeled with their
corresponding number or Greek letter.
In this work, the refined x-ray crystallographic structures of
selenomethionyl CMP-NeuAc synthetase and CMP-NeuAc synthetase in
complex with the substrate analogue CDP (CMP-NeuAc synthetase-CDP) are
reported. The active site and substrate binding sites for this class of
enzyme are described in detail for the first time, and a comparison of
the two structures indicates substrate binding is accompanied by
significant conformational rearrangements within the active site.
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EXPERIMENTAL PROCEDURES |
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Protein Purification--
Recombinant CMP-NeuAc synthetase was
overexpressed in E. coli and purified using published
protocols (18) with modifications. Briefly we used a combination of
anion-exchange on a MonoQ column (Amersham Pharmacia Biotech) and gel
filtration on a Superose 12 column (Amersham Pharmacia Biotech).
Selenomethionyl CMP-NeuAc synthetase was overexpressed in a methionine
auxotroph of E. coli (WA-834) grown in a defined medium
containing 40 mg liter1 selenomethionine (19) and
purified using the same protocol. All protein samples were purified to
homogeneity and were of the expected molecular mass as judged by mass
spectrometry and SDS-polyacrylamide gel electrophoresis.
Crystallization and Data Collection--
All crystals used in
this work were grown by vapor diffusion using hanging drops consisting
of 3 µl of protein stock (5.0 mg ml1 in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol, and 0.1 mM EDTA) and 3 µl
of reservoir solution. Crystals of CMP-NeuAc synthetase and CMP-NeuAc
synthetase-CDP were both grown over reservoir solutions composed of
16% polyethylene glycol 2000, 100 mM Tris-HCl, pH 8.0, 10 mM MgCl2 and 1 mM dithiothreitol.
The CMP-NeuAc synthetase crystals grow in 2-3 days, are orthorhombic
(space group, P212121; unit cell,
a = 42.99, b = 59.52, c = 157.56 Å;
=
=
= 90°), and a 2.0-Å resolution data set has been
collected at Brookhaven National Laboratory in the Biology Department
single-crystal diffraction facility at beamline X8-C in the National
Synchrotron Light Source. Orthorhombic crystals of CMP-NeuAc
synthetase-CDP were grown in the presence of 10 mM CDP
(unit cell, a = 43.01, b = 69.89, c = 157.78 Å;
=
=
= 90°), appeared after 7 days, and
diffracted to 2.2 Å on a local Rigaku RU200 rotating anode (50 kV, 100 mA) equipped with OSMIC mirrors. All CMP-NeuAc synthetase
crystals in this work were rod-shaped and the largest crystals grew to ~0.80 × 0.15 × 0.15 mm in size. All intensity data sets
were collected at 100 K after flash-freezing in mother liquor
containing 22% glycerol. Intensity data collection and processing
statistics for each data set used in this work are summarized in Table
I.
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Structure Solution-- The structure of the CMP-NeuAc synthetase homodimer was determined by MAD phasing based on 10 selenium atom positions. The Se atom positions were identified using both SHAKE'N'BAKE (20) and SOLVE (21) and subjected to positional, isotropic B-factor, and occupancy refinement within SOLVE prior to the calculation of phases (Table I). The initial phase estimates were improved by density modification using DM (22) prior to calculation of a 2.0-Å MAD phased electron density map. The initial electron density map is easily interpretable and an initial trace of the CMP-NeuAc synthetase main chain was generated within XTALVIEW (23). The CMP-NeuAc synthetase-CDP structure was determined by molecular replacement using the CMP-NeuAc synthetase model and the program AMORE (24). Both, CMP-NeuAc synthetase2 and CMP-NeuAc synthetase-CDP3 were refined using CNS 1.0 (25), and all interactive fitting of the models made use of XTALVIEW. Table II lists common statistical indicators for both refined models.
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Structure Analysis and Figure Preparation--
PROMOTIF (26) and
PROCHECK (27) were used to identify secondary structures and to check
the stereochemical quality of both models throughout the refinement.
SURFACE (22) and GRASP (28) were utilized to examine the molecular
surface of the models and to examine the local environment of specific
residues. XTALVIEW was used for various least-squares superpositions
and DOCKVISION (29) was used for the NeuAc modeling. Docking made use
of an energy-minimized acylneuraminate structure derived from Protein Data Bank accession code 1WIA and the refined CMP-NeuAc synthetase-CDP model. The starting NeuAc model was used to generate all common NeuAc
conformations, and all conformations were docked (10,000-50,000 trials) against a static CMP-NeuAc synthetase-CDP model using either an
energy term or conformational energy terms as a scoring function. The
best docking results were obtained using CMP-NeuAc synthetase-CDP
and CDP conformation I. The figures were prepared using
GRASP (28) and MOLSCRIPT (30).
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RESULTS AND DISCUSSION |
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Overall Fold--
The overall fold of the CMP-NeuAc synthetase-CDP
homodimer is presented in Fig. 2. The
CMP-NeuAc synthetase monomer is composed of an ~190 residue, globular
/
domain (~50 × 50 × 40 Å) that is structurally
classified as an
, three-layered sandwich (31) and a small
35-residue domain that is directed away from the rest of the monomer
and forms an extended dimerization interface. The dimerization domains
of each monomer wrap around one another in a tight interaction and make
contacts with the globular
/
domain of the opposite monomer. The
resulting functional homodimer is highly asymmetric with dimensions of
~85 × 45 × 40 Å.
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A twisted, central -sheet forms the hydrophobic core of the
/
domain of the monomer and is flanked by helices giving rise to the
, three-layered sandwich structure. The
-sheet contains six
parallel
strands (
1,
4,
5,
6,
7,
11) and a single antiparallel
strand (
10) connected with a
1x,
1x, +3x, +2x,
1x, +2x topology (32). The connectivity of consecutive
parallel
strands is right-handed except for a relatively rare
left-handed linkage between strand
6 and strand
7. Alignments of
known CMP-NeuAc synthetase sequences (33) indicate that residues
between strands
6 and
7 (102) are among the most highly
conserved in the comparison (Fig. 3). Three
helices (B, C, F) pack
against and bury the face of the central
-sheet furthest from the
dimerization interface. The remaining face of the
-sheet is covered
by two
helices (E, H) and contacts with the dimerization domain of
the opposite monomer. The 310 helix A, a short
ribbon
(
2,
3), 310 helix D, and
helix I are all located
on the C-terminal side of the mostly parallel, central
-sheet. These
secondary structures and their associated loops form a deep pocket at
the C-terminal ends of the
-sheet that is the mononucleotide-binding
pocket. The presence of a substrate-binding pocket at the C-terminal
ends of predominantly parallel
-sheets is a recurring theme in
structural biology (34).
The dimerization domain contains a 310 helix (helix G) and
a ribbon (strand
8 and strand
9) that is continuous with the central
-sheet of the opposite monomer. The dimerization domain is
located between strands
7 and
10 and is directed away from the
rest of the
/
domain by a
-bulge at position 175 of strand
10. In the homodimer, a
-bulge in strand
10 facilitates an antiparallel hydrogen-bonding network between the N terminus of strand
10 and strand
8 of the opposite monomer. The dimerization interface buries a large surface area despite its modest size. Using a
probe radius of 1.4 Å, a single monomer buries ~1450 Å2
in the dimer (35).
A search of representative structures in the Protein Data Bank using
the DALI server (36) indicates the CMP-NeuAc synthetase overall fold
has only limited homology with other known folds in the Protein Data
Bank. In general, enzymes containing a mononucleotide-binding (Rossman)
fold (37) resemble the N-terminal 100 residues of the /
domain,
up to but not encompassing the left-handed linkage between strands
6
and
7. The similarities between the N terminus of CMP-NeuAc
synthetase and Rossman fold containing proteins are restricted to the
order and connectivity of secondary structures, because CMP-NeuAc
synthetase and related enzymes have unique mononucleotide-binding pockets. The CMP-NeuAc synthetase
/
domain fold is similar to that reported for
cytosine-5'-monophosphate-2-keto-3-deoxy-manno-octonate synthetase (CMPKDOS (38)), a homologous, dimeric enzyme that synthesizes a related, activated sugar compound,
cytosine-5'-monophosphate 2-keto-3-deoxy-manno-octonate (CMP-KDO).
These two enzymes are similar in length but share limited amino acid
sequence identity (18%, Fig. 3), and the
fold of the CMPKDOS dimerization domain is significantly different from
the equivalent structure in this work. In the CMPKDOS monomer, the
dimerization domain is globular and compact and does not make contacts
with the
/
domain of the opposite monomer. Despite the
differences in the dimerization interface, both enzymes share a highly
asymmetric overall shape. Because the CMPKDOS coordinates are yet to be
released and were not made available to us, we cannot present a
detailed comparison of the CMP-NeuAc synthetase and CMPKDOS overall
folds.
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Comparison of CMP-NeuAc Synthetase and CMP-NeuAc
Synthetase-CDP--
The two monomers of CMP-NeuAc synthetase-CDP in
the asymmetric unit have nearly identical main-chain conformations. The
monomers superpose with an r.m.s. deviation of 0.60 Å when considering all common main-chain atoms (888 of 912 atoms). Similarly, there are no
large-scale conformational differences in a comparison of the CMP-NeuAc
synthetase- and CMP-NeuAc synthetase-CDP-refined models and the two
homodimers superpose with an r.m.s. deviation of 0.71 Å for 1728 equivalent main-chain atoms (Fig. 4). The
most prominent differences in a comparison of the two refined models occur in the mononucleotide-binding pocket. In the unliganded CMP-NeuAc
synthetase structure, the P-loop (residues 10-22) and residues
following strand 5 (71) have relatively weak electron density or
are fully disordered in the refined maps. In the CMP-NeuAc synthetase-CDP structure, the equivalent residues make specific contacts with the bound substrate analogue (Table
III and see Fig. 6) either directly or
through ordered solvent molecules, and have well ordered electron
density in the refined maps. The average main-chain temperature factors
for residues 10-22 and 71-80 of CMP-NeuAc synthetase-CDP and
CMP-NeuAc synthetase are 33.1 Å2 (184 atoms) and 50.3 Å2 (128 atoms), respectively. In CMP-NeuAc synthetase-CDP,
the P-loop primarily interacts with the ribose and phosphate moieties
of the substrate analogue, whereas residues 71-80 are responsible for
base recognition. At the same time, these residues make relatively few
specific contacts with the remainder of the enzyme. Apparently, CDP
itself provides specific contacts that allow these residues to fold
about the mononucleotide substrate analogue and become relatively
ordered. Consequently, we propose mononucleotide binding is accompanied
by a conformational rearrangement that orders both the P-loop and
residues 71-80. This model of substrate binding can account for the
observed differences in the refined electron density of the two
structures and the observed depth of the mononucleotide-binding pocket.
It also explains the ordered bi bi kinetic mechanism (CTP binds first
and CMP-NeuAc dissociates last) observed in the homologous enzyme from
H. ducreyi (39). The P-loop and residues 71-80 contain 7 of
the 13 invariant residues in an alignment of known bacterial CMP-NeuAc
synthetase enzymes (Fig. 3). In agreement with this work, amino acid
sequence analysis, site-directed mutagenesis, and chemical modification
studies indicate residues 10-22 (phosphate binding loop or P-loop)
following strand
1 are integral components of the
mononucleotide-binding pocket (33, 40). Likewise, in the CMPKDOS
structure (38), residues of the P-loop were observed to bind a heavy
atom derivative (IrCl
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Mononucleotide Binding Pocket--
The molecular surface of
CMP-NeuAc synthetase in the presence of CDP is shown in Fig.
5. The large cleft at the C-terminal end
of the central -sheet is the mononucleotide-binding pocket. The
cleft is formed by residues following strand
1 (P-loop), strand
5
(71), and strand
11 (208). Polar residues line the cleft
and are responsible for binding the nucleotide substrate (Fig.
6 and Table III). As shown in Fig.
7, the substrate analogue, CDP, is bound
in the mononucleotide-binding pocket in two distinct conformations. The
cytosine base and ribose moiety are equivalent in the two
conformations, whereas the
and
phosphoryl groups adopt
different conformations (I, II) as a result of differences in the
C4'-C5'-O5'-P1A and C5'-O5'- P1A-O3A torsion angles.
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The cytosine base is completely buried (521 Å2) in the
deep cleft, and its position is fixed by a series of hydrogen bonds. The N1 and N
2
atoms of the invariant Arg-71 donate hydrogen bonds to O2
and N3 of the base and give rise to the enzymes specificity for
cytosine nucleotides. Arg-12 N also donates a hydrogen bond to O2 while Ala-80 O and an ordered solvent molecule accept hydrogen bonds from N4.
These interactions completely satisfy the hydrogen bonding potential of
the base. The cytosine base orientation is further fixed by van der
Waals contacts with the Leu-10 side chain and a stacking interaction
with the Arg-12 guanidinium. Together, the Leu-10 and Arg-12 side
chains form a narrow pocket that contacts both faces of the base. The
residues that bind or contact the cytosine base are all derived from
the P-loop and residues following strand
5 (71). These are the
same regions that are largely disordered in the CMP-NeuAc synthetase
structure determined in the absence of a substrate analogue. The
hydrogen bonding potential of the ribose O2' and O3' atoms are
satisfied by Asn-22 N
2 and
O
1, respectively. Residues Gln-104, Pro-105,
and Thr-106 form the remainder of the ribose subsite of the
mononucleotide-binding pocket and are part of the conserved left-handed
linkage between strand
6 and strand
7. Leu-10, Arg-12, and
several ordered solvent molecules also contribute to the ribose
subsite. Residues of the P-loop are the most highly conserved in
sequence alignments of bacterial CMP-NeuAc synthetase enzymes and
residues Arg-12, Ser-15, Lys-16, Lys-21, and Asn-22 are invariant in
these alignments (Fig. 3).
Notably, Lys-16 and Asn-22 are not conserved in CMPKDOS sequences. Whereas it has been demonstrated that Lys-16 is not essential for CMP-NeuAc synthetase activity (40), catalysis is more efficient in the Lys-16-containing holoenzyme. The lack of conservation of Asn-22 is more difficult to understand. In CMP-NeuAc synthetase, Asn-22 hydrogen bonds to the O2' and O3' atoms of the ribose moiety and apparently discriminates between CTP and dCTP. CMPKDOS has a proline residue at the equivalent position in amino acid sequence alignments. Clearly, proline is unable to specifically interact with the ribose in a manner similar to that observed in CMP-NeuAc synthetase. In the absence of a description of the CMPKDOS active site it is unclear how CMPKDOS specifically binds the ribose moiety and discriminates between CTP and dCTP.
The two conformations of the and
phosphoryl groups of the
substrate analogue CDP are a result of differences in the torsion angles about the C5'-O5' and O5'-P1A bonds (Fig. 7), respectively. They
have been modeled as equivalent conformations during the refinement and
have comparable average isotropic B-factors. Conformation I
of the
and
phosphoryl group has a C4'-C5'-O5'-P1A torsion angle
of
115° and a C5'-O5'-P1A-O3A torsion angle 155°, whereas conformation II has torsion angles of
160° and 50°, respectively. Several observations strongly suggest that conformation I closely mimics that of the substrate, CTP: (a) the presence of an
oxyanion-hole-like structure formed by Ser-15 N and Gly-17 N that is
adjacent to the
phosphate and is suitably positioned to interact
with a
phosphate, (b) the presence of a potential
Mg2+-binding site bridging the phosphoryl groups of the
substrate and including the relatively conserved Asp-209 and Asp-211,
and (c) the number and nature of the contacts between
CMP-NeuAc synthetase and the substrate analogue phosphoryl groups
(Table III and Fig. 6). Additionally, in conformation I a single,
ordered water molecule (Wat-160) hydrogen bonding to Asn-14 O and the
phosphate, is suitably positioned to donate a hydrogen to the
pyrophosphate leaving group. In CDP conformation II, there are no
apparent
phosphate- or Mg2+-binding sites and
relatively fewer contacts between the enzyme and phosphoryl
groups. Why then do we observe conformation II in this work? The
C4'-C5'-O5'-P1A dihedral angle (conformation II) adopts a staggered
conformation and relieves the stereochemical strain arising from the
eclipsed C4'-C5'-O5'-P1A dihedral angle present in conformation I. The
absence of a binding site for the
phosphate in conformation II
suggests CTP will not bind in two distinct, equally populated
conformations as observed with CDP.
The bulk of the interactions with phosphate moieties, in conformation
I, are from residues of the P-loop. This is similar to the case for
many Rossman fold proteins, which also have a phosphate binding or
P-loop immediately following their N-terminal strand. However, the
details of the interaction between the P-loop and the base and
phosphate are very different in these two groups of enzymes. This is
not surprising because CMP-NeuAc synthetase releases pyrophosphate as a
product, whereas kinases, G-proteins, and other Rossman fold-containing
proteins release a single phosphate, the
phosphoryl group (37). In
conformation II, there are fewer contacts between the phosphates and
the protein.
NeuAc Binding Pocket-- As seen in Fig. 5, the active site cleft of CMP-NeuAc synthetase extends beyond the mononucleotide-binding pocket toward the dimerization domain and forms the NeuAc-binding pocket. The pocket is lined with a number of polar side chains (Fig. 5, 6) and can easily accommodate NeuAc. Atoms O4 and O9 and the NeuAc carboxylate are solvent-exposed and may interact with ordered solvent molecules in addition to CMP-NeuAc synthetase. Alternatively, substrate binding may involve a small conformational change in CMP-NeuAc synthetase that decreases the volume of the binding pocket. The polar side chains of residues Ser-82, Gln-104, Thr-106, and Asp-209 line the NeuAc pocket nearest the bound substrate analogue (CDP), whereas Tyr-179 and residues Glu-137, Lys-142, Glu-162, and Arg-165 from the dimerization domain of the opposite monomer form the edges of the binding pocket furthest from the bound CDP. Interestingly, 4 of the 9 polar residues within the proposed NeuAc-binding pocket are derived from residues of the dimerization domain. This implies that the sequence and structure of the dimerization domain is an important determinant for the discrimination of various sialic acids. Thus, the dimerization domains of homologous enzymes with specificities for related sialic acid derivatives are likely to have limited amino acid sequence identity and adopt a conformation different from that observed in CMP-NeuAc synthetase. As pointed out earlier, the dimerization interfaces of CMP-NeuAc synthetase and CMPKDOS are very different, and sequence alignments indicate the dimerization domains of CMP-NeuAc synthetase enzymes are not highly conserved (35, Fig. 3).
NeuAc Modeling--
A model of an energy-minimized NeuAc that has
been docked in the NeuAc-binding pocket is presented in Fig.
8. The docked NeuAc model produces the
lowest energy using either the DockVision energy function (31.04 kJ)
or conformational energy function (
9.40 kJ). In the docked model, the
NeuAc functional groups make a number of plausible interactions with
invariant or conserved residues (Figs. 6 and 8). The axial, O2 atom of
NeuAc is directed at the
phosphate of the biologically significant
CDP conformer (conformation I) in accordance with the known specificity
of CMP-NeuAc synthetase (41). The NeuAc C2 carboxylate (a strong acid)
is directed away from the cleft and forms a salt bridge with the
Arg-165 guanidinium in the model. As seen in Fig. 3, a positively
charged amino acid able to counter the negative charge of the C2
carboxylate is present at positions 164 or 165 of each amino acid
sequence. The N-acetyl group at C5 of NeuAc docks with the
acetyl oxygen exposed to bulk solvent, the nitrogen atom hydrogen
bonding with Gln-104 O
and the methyl group packing
against the aromatic residues Tyr-179, Phe-192, and Phe-193. Although,
these residues are all conserved among CMP-NeuAc synthetase enzymes
(Fig. 3), only Gln-104 is conserved between CMP-NeuAc synthetase and
CMPKDOS enzymes. This is reasonable because KDO, the CMPKDOS substrate,
has a hydroxyl at position C5 (equivalent to the nitrogen of NeuAc) but
has no counterpart to the carbonyl oxygen and methyl group of NeuAc.
Consequently, only the Gln-104 interaction with the C5 substituent is
expected to be conserved between these enzymes.
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The O4 atom of NeuAc is paired with the Ser-82 OH in this model and may also interact with the Ala-80 N. At the base of the binding pocket, the Gln-104 side chain contacts both the O8 and N5 atoms of NeuAc model and is suitably positioned to help CMP-NeuAc synthetase discriminate between the sialic acids with variable C6 functional groups. The O8 atom is also within contact distance of the Asp-209 carboxylate. The O7 atom and O9 atoms of NeuAc are directed at the dimerization domain of the opposite monomer and are within hydrogen-bonding distances of the Lys-142 and Tyr-179 side chains. Additional residues may interact with the NeuAc model via bridging solvent molecules or may directly interact with NeuAc if CMP-NeuAc synthetase undergoes a small conformational change upon substrate binding.
In the CMP-NeuAc synthetase reaction mechanism, the O2 atom of NeuAc is
the nucleophile in an SN2 attack on the phosphate (41).
Our structure and docking results support this mechanism and we predict
the leaving group pyrophosphate is stabilized by the absolutely
conserved Arg-12, Lys-21, one or more bridging Mg2+
cations, and possibly the conserved Lys-16 (Fig. 3). As previously mentioned, an ordered solvent molecule hydrogen bonding to Asn-14 O is
suitably positioned to donate a hydrogen bond to the leaving group
pyrophosphate. In the model, the nucleophilic NeuAc O2 atom is not
within hydrogen-bonding distance of any atom of the enzyme and,
consequently, there is no readily apparent general base. An ordered
solvent molecule may serve as the general base or, alternatively, a
conformational rearrangement may bring a general base within
hydrogen-bonding distance of the O2 atom upon NeuAc binding. We favor a
mechanism in which an ordered solvent molecule serves as the general
base, because it seems unlikely that a conformational rearrangement
could bring a protein atom within hydrogen-bonding distance of the
NeuAc O2 atom without disrupting the specific interactions detailed above.
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ACKNOWLEDGEMENTS |
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We thank M.-F. Karwaski for technical help in the purification of the CMP-NeuAc synthetase and the selenomethionyl CMP-NeuAc synthetase, D. Krajcarski for mass spectrometry measurements, and the instructors and staff at the National Synchrotron Light Source Data Col'99 course for their generous assistance. This work was supported by an NSERC strategic grant (to W. W. and N. S.). S. M. is a NSERC post-doctoral fellow. N. S. is a Canadian Institute of Health Research Scholar, a Howard Hughes Medical Institute International Scholar, and a Burroughs Wellcome New Investigator.
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FOOTNOTES |
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* 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.
§ Present address: Dept. of Chemistry and Biochemistry, University of Lethbridge, 4401 University Dr., Lethbridge T1K 3M4, Canada.
To whom correspondence should be addressed: Tel.:
604-221-0789; Fax: 604-221-5227; E-mail:
natalie@byron.biochem.ubc.ca.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M007235200
2 The atomic coordinates for CMP-NeuAc synthetase are available in the Protein Data Bank under PDB 1EYR.
3 The atomic coordinates for CMP-NeuAc synthetase-CDP are available in the Protein Data bank under PDB 1EZI.
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ABBREVIATIONS |
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The abbreviations used are: CMP-NeuAc synthetase, CMP-acylneuraminate synthetase or cytosine-5'-monophosphate-N-acetyl neuraminic acid synthetase; CMP-NeuAc, cytosine-5'-monophosphate-N-acetyl neuraminic acid; MAD, multiple wavelength anomalous dispersion; NeuAc, acylneuraminate; CMPKDOS, cytosine-5'-monophosphate-2-keto-3-deoxy-manno-octonate synthetase; KDO, 2-keto-3-deoxy-manno-octonate; r.m.s., root mean square.
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