Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP*

Steven C. MosimannDagger §, Michel Gilbert, Dennise DombroswkiDagger , Rebecca To, Warren Wakarchuk, and Natalie C. J. StrynadkaDagger ||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.



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Fig. 1.   The reaction catalyzed by CMP-NeuAc synthetase in the presence of Mg2+. CMP-NeuAc synthetase specifically utilizes the beta  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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 liter-1 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 ml-1 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; alpha  = beta  = gamma  = 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; alpha  = beta  = gamma  = 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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Table I
Summary of data collection and MAD phasing statistics for CMP-NeuAc synthetase-CDP and CMP-NeuAc synthetase

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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Table II
Summary of refinement statistics for selenomethionyl CMP-NeuAc synthetase and CMP-NeuAc synthetase in complex with CDP

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).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha /beta domain (~50 × 50 × 40 Å) that is structurally classified as an alpha beta alpha , 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 alpha /beta domain of the opposite monomer. The resulting functional homodimer is highly asymmetric with dimensions of ~85 × 45 × 40 Å.



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Fig. 2.   The CMP-NevAc synthetase homodimer in complex with CDP. CMP-NevAc synthetase is shown as a barrel (helices) and arrow (strands) drawing, and the CDP substrate analogue is shown as a van der Waals surface. Monomer A is indicated with dark gray helices and strands, while monomer B is indicated with light gray helices and strands. CMPANS has a single alpha /beta domain with an alpha beta alpha three-layered sandwich fold and 35 residue protrusion that forms the dimerization interface. The 11 beta  strands (arrows) are arranged in a 7-stranded, mostly parallel beta -sheet and two beta  ribbons. The eight helices pack against either side of the central beta -sheet or form part of the dimerization interface (helix G).

A twisted, central beta -sheet forms the hydrophobic core of the alpha /beta domain of the monomer and is flanked by helices giving rise to the alpha beta alpha , three-layered sandwich structure. The beta -sheet contains six parallel beta  strands (beta 1, beta 4, beta 5, beta 6, beta 7, beta 11) and a single antiparallel beta  strand (beta 10) connected with a -1x, -1x, +3x, +2x, -1x, +2x topology (32). The connectivity of consecutive parallel beta  strands is right-handed except for a relatively rare left-handed linkage between strand beta 6 and strand beta 7. Alignments of known CMP-NeuAc synthetase sequences (33) indicate that residues between strands beta 6 and beta 7 (102) are among the most highly conserved in the comparison (Fig. 3). Three alpha  helices (B, C, F) pack against and bury the face of the central beta -sheet furthest from the dimerization interface. The remaining face of the beta -sheet is covered by two alpha  helices (E, H) and contacts with the dimerization domain of the opposite monomer. The 310 helix A, a short beta  ribbon (beta 2, beta 3), 310 helix D, and alpha  helix I are all located on the C-terminal side of the mostly parallel, central beta -sheet. These secondary structures and their associated loops form a deep pocket at the C-terminal ends of the beta -sheet that is the mononucleotide-binding pocket. The presence of a substrate-binding pocket at the C-terminal ends of predominantly parallel beta -sheets is a recurring theme in structural biology (34).

The dimerization domain contains a 310 helix (helix G) and a beta  ribbon (strand beta 8 and strand beta 9) that is continuous with the central beta -sheet of the opposite monomer. The dimerization domain is located between strands beta 7 and beta 10 and is directed away from the rest of the alpha /beta domain by a beta -bulge at position 175 of strand beta 10. In the homodimer, a beta -bulge in strand beta 10 facilitates an antiparallel hydrogen-bonding network between the N terminus of strand beta 10 and strand beta 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 alpha /beta domain, up to but not encompassing the left-handed linkage between strands beta 6 and beta 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 alpha /beta 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 alpha /beta 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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Fig. 3.   Amino acid sequence alignment of the substrate binding regions of known bacterial CMP-NeuAc synthetase enzymes and a representative bacterial CMPKDOS. Shaded boxes indicate amino acid identity, unshaded boxes indicate conservative amino acid replacements and labels indicate the role of each region in substrate binding. The equivalent residues from a representative CMPKDOS sequence are shown below and the residue numbers are from the N. meningitidis enzyme. HI (H. influenza), HD (H. ducreyi), NM (N. meningitidis), AC (Aeromonas caviae), CC (Campylobacter coli), EC (E. coli), SA (Streptococcus agalactiae), HP (Helicobacter pylori), KDS (CMPKDOS from H. influenzae).

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 beta 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 beta 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<UP><SUB>6</SUB><SUP>3−</SUP></UP>), and the authors speculated that these residues play a role in phosphate binding.



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Fig. 4.   CMP-NeuAc synthetase active site in the presence and absence of CDP. The CMP-NeuAc synthetase-CDP (blue strands and green helices) and CMP-NeuAc synthetase (dark orange strands and red helices) structures superpose with a r.m.s. deviation of 0.71 Å for all common main-chain atoms, and the only relatively, large conformation differences are associated with residues implicated in mononucleotide binding. In the presence of CDP, the P-loop and helix D (light green ribbon) are well ordered and directly contact the bound substrate analogue. In the absence of CDP, the equivalent residues are poorly ordered (P-loop) or disordered (helix D). This suggests substrate binding is accompanied by conformational rearrangements that orders residues involved in mononucleotide binding.


                              
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Table III
Hydrogen bonds and van der Waals contacts between CMP-NeuAc synthetase and CDP

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 beta -sheet is the mononucleotide-binding pocket. The cleft is formed by residues following strand beta 1 (P-loop), strand beta 5 (71), and strand beta 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 alpha  and beta  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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Fig. 5.   Molecular surface of the CMP-NeuAc synthetase active site. Monomer A is shown as a GRASP surface colored according to its calculated electrostatic potential (red for negative and blue for positive). Monomer B is shown as Calpha trace (light blue tube). The CDP-binding pocket is deep and lined with polar residues from the P-loop (residues 10-22) and loops on either side of helix D (residues 70-72 and 76-82). Adjacent to the CDP-binding pocket is the NeuAc-binding pocket, a second deep cleft comprising residues from both monomers of the CMP-NeuAc synthetase homodimer. In particular, Ser-82, Gln-104, Thr-106, and Tyr-179 of monomer A and Lys-142 and Arg-165 of monomer B line the NeuAc-binding pocket.



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Fig. 6.   Observed and modeled contacts between CMP-NeuAc synthetase and CDP and CMP-NeuAc synthetase and NeuAc, respectively. A, observed hydrogen bonds and contacts between CMP-NeuAc synthetase and CDP. Hydrogen bonds are indicated with solid lines and residues providing contacts are listed adjacent to the atoms they contact. Arrows indicate donated hydrogen bonds as inferred from the structure. Two ordered, solvent molecules form hydrogen bonds with CDP and are indicated as circles. B, modeled hydrogen bonds (dashed lines) and contacts from the docking (29) of NeuAc with CMP-NeuAc synthetase-CDP. The model suggests a number of plausible interactions. Residues at positions 82, 104, 106, 142, 165, 179, 193, and 209 are conserved or conservatively replaced in CMP-NeuAc synthetase sequences from Neisseria, Haemophilus, Aeromonas, and Campylobacter species (~230 residues). The equivalent residues, excepting 142, are conserved in the larger CMP-NeuAc synthetase enzymes of Escherichia, Helicobacter, and Streptococcus species.



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Fig. 7.   The refined 2Fo - Fc electron density associated with the bound CDP substrate analogue. The CDP-binding site of CMP-NeuAc synthetase-CDP is shown as a barrel and strand drawing while residues directly contacting the bound CDP and the two observed CDP conformations are indicated in ball-and-stick. The two diphosphate conformations of CDP are labeled I (green phosphorous atoms) and II (yellow phosphorous atoms). The biologically significant conformation of the alpha  and beta  phosphoryl groups is conformation I (green phosphorous atoms). The refined 2Fo - Fc electron density for the bound CDP is contoured at 1.2 sigma .

The cytosine base is completely buried (521 Å2) in the deep cleft, and its position is fixed by a series of hydrogen bonds. The Neta 1 and Neta 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 beta 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 Ndelta 2 and Odelta 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 beta 6 and strand beta 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 alpha  and beta  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 alpha  and beta  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 beta  phosphate and is suitably positioned to interact with a gamma  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 beta  phosphate, is suitably positioned to donate a hydrogen to the pyrophosphate leaving group. In CDP conformation II, there are no apparent gamma  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 gamma  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 beta  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 gamma  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 alpha  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 Oepsilon 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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Fig. 8.   A model of NeuAc (yellow carbons) docked in the NeuAc binding cleft and its relation to the bound CDP (black carbons). In the lowest energy docking, the NeuAc carboxylate is directed away from the cleft and the O4 and N-acetyl functional groups are directed into the cleft. The hydroxyl groups, O7, O8, and O9 of NeuAc are within hydrogen-bonding distance of Lys-142 (monomer B), Asp-209 and Tyr-179, respectively. The view has been chosen to show the contacts between the modeled NeuAc and CMP-NeuAc synthetase. The nucleophilic O2 atom (hidden under the NeuAc carboxylate) is directed at the alpha  phosphate of CDP and is positioned for an SN2 attack. There are no protein atoms suitably positioned to act as a general base, and an ordered solvent molecule likely serves as the general base in this mechanism.

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 alpha  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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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