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J. Biol. Chem., Vol. 277, Issue 6, 4343-4350, February 8, 2002

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Structure and Mechanism of CTP:Phosphocholine Cytidylyltransferase (LicC) from Streptococcus pneumoniae^*

Bo-Yeon Kwak, Yong-Mei Zhang^§, Mikyung Yun, Richard J. Heath^§, Charles O. Rock^§¶, Suzanne Jackowski^§¶, and Hee-Won Park^¶

From the  Department of Structural Biology and the ^§ Protein Science Division, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the ^¶ Department of Molecular Biosciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, September 21, 2001, and in revised form, November 8, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pneumococcal LicC is a member of the nucleoside triphosphate transferase superfamily and catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline. The structures of apo-LicC and the LicC·CDP-choline·Mg²⁺ ternary complex were determined, and the comparison of these structures reveals a significant conformational change driven by the multivalent coordination of Mg²⁺. The key event is breaking the Glu²¹⁶·Arg¹²⁹ salt bridge, which triggers the coalescence of four individual -strands into two extended -sheets. These movements reorient the side chains of Trp¹³⁶ and Tyr¹⁹⁰ for the optimal binding and alignment of the phosphocholine moiety. Consistent with these conformational changes, LicC operates via a compulsory ordered kinetic mechanism. The structures explain the substrate specificity of LicC for CTP and phosphocholine and implicate a direct role for Mg²⁺ in aligning phosphocholine for in-line nucleophilic attack and stabilizing the negative charge that develops in the pentacoordinate transition state. These results provide a structural basis for assigning a specific role for magnesium in the catalytic mechanism of pneumococcal LicC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Choline metabolism plays a key role in cell separation, transformation, autolysis, and pathogenicity of Streptococcus pneumoniae. The only known metabolic fate of choline is to decorate the teichoic and lipoteichoic acids of the cell wall, and choline is an essential nutrient for S. pneumoniae (1). The cell surface P-Cho¹ facilitates the interaction with the host surface and induces attachment and invasion (2, 3). The importance of choline in pathogenesis is not confined to S. pneumoniae but also plays a role in Hemophilus influenzae (4-7), Pseudomonas aeruginosa, and Neisseria gonorrhoeae (8, 9). In addition, cell wall P-Cho serves as the scaffold for a group of choline-binding proteins that are secreted from the cells and subsequently attached to the cell surface by their homologous choline-binding domains (see Ref. 10 and references therein). These choline-binding proteins are essential for many aspects of S. pneumoniae cell physiology including competence and stationary phase lysis.

The pathway for choline metabolism in S. pneumoniae and H. influenzae has been hypothesized to consist of a choline transport system, a choline kinase, CTP:phosphocholine cytidylyltransferase (CCT), and a choline phosphotransferase that transfers P-Cho from CDP-Cho to either lipoteichoic acid or lipopolysaccharide (5). The existence of this pathway is supported by the detection of choline kinase and CCT activity in crude extracts of S. pneumoniae (11, 12). Genetic elements required for choline incorporation into the lipopolysaccharide of H. influenzae are found in the lic1 locus, which contains four open reading frames. The hypothesis drawn from the bioinformatic analysis of the lic1 locus (4) is that licA corresponds to choline kinase based on a 31% identity to the choline kinase of Saccharomyces cerevisiae over the short span of 40 amino acids between residues 222 and 262. The licB gene has several predicted transmembrane domains and is thus postulated to be a choline transporter. The hydrophilic licC gene product is a candidate for the CCT due to the resemblance of its amino terminus to the amino-terminal 60 residues of NTP transferase family members, leaving the licD gene as a candidate for the choline phosphotransferase. A homologous licC gene exists in S. pneumoniae (13), and the predicted LicC proteins of H. influenzae and S. pneumoniae are 37% identical and 60% similar.

Recently, the LicC of S. pneumoniae was purified and demonstrated to catalyze the CCT reaction (14). The enzyme possesses a high degree of selectivity for P-Cho and CTP, although phosphoethanolamine and ATP are poor substrates. LicC lacks homology to either the prototypical metazoan CCTs (15, 16) or the CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis (17). Rather, LicC is related to members of the NTP transferase superfamily that primarily activate sugars by transferring the NMP moiety to a phosphorylated acceptor substrate concomitant with the release of PP_i (see the NCBI Conserved Domain Database, protein family 00483) and contain the N-terminal signature sequence KAX₅GXGTRX_6-9K. The inclusion of LicC in this family is based on its similarity to the amino termini of Escherichia coli GalU and P. aeruginosa RmlA, two prototypical nucleotidyltransferases that form NDP-hexose derivatives that are used in the biosynthesis of bacterial cell wall components (14). Members of this superfamily are characteristically dimers or tetramers; however, LicC is a monomer (14).

The crystal structures of three members of the NTP transferase superfamily are known. These are the dimeric GlmU (18) and methylerythritol-4-phosphate cytidylyltransferase from E. coli (19) and the tetrameric RmlA from both P. aeruginosa (20) and Salmonella enterica (21). The NMP transferase domains of these enzymes have a similar overall folding, and they share several key residues involved in substrate binding and/or catalysis, such as Gly¹⁴, Arg¹⁸, and Lys²⁸ (LicC numbering) in the signature sequence. Magnesium is required for activity. In S. enterica RmlA and E. coli methylerythritol-4-phosphate cytidylyltransferase, Mg²⁺ is proposed to position the nucleotide substrate during the NMP transfer reaction and to stabilize the PP_i leaving group after the reaction (19, 21); thus, Mg²⁺ is not thought to play a role in the catalytic mechanism. In the other two structures, the magnesium binding site is undefined (18, 20). Several positively charged residues that are not conserved in all of the NTP transferase structures are proposed to be involved in catalysis of RmlA and methylerythritol-4-phosphate cytidylyltransferase by providing a favorable electrostatic environment, although their exact role is unclear (19-21). Exceptions are Arg¹⁸ and Lys²⁸ (LicC numbering) in the signature sequence that bind the triphosphate group of the nucleotide substrate to stabilize the negative charge developed on the oxygens of the -phosphate in the pentacoordinate transition state and the released PP_i product. We have determined the kinetic mechanism of LicC from S. pneumoniae and the structures of LicC and the LicC·CDP-Cho·Mg²⁺ ternary complex. The structures explain the specificity of LicC for CTP and identify the active site residues involved in binding the product, CDP-choline, with magnesium. We propose the mode of substrate binding and the direct role of the magnesium in catalysis based on the kinetic data and structures.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Sources of supplies were Sigma (for CTP, CDP-Cho, P-Cho, and seleno-L-methionine) and Amersham Biosciences, Inc. (for phospho[methyl-¹⁴C]choline (specific activity 55 mCi/mmol)). His-tagged native LicC was expressed and purified as described previously (14). For metabolic incorporation of selenomethionine into LicC, pET-15b containing the licC gene was transformed into BL21-CodonPlus(DE3)-RIL-X (Stratagene), and protein was expressed as described previously (22) and purified in the same manner as the His-tagged native LicC. Protein concentration was determined by the method of Bradford (23). All other chemicals were reagent grade or better.

LicC Enzyme Assay-- The enzymatic activity of LicC under different conditions was studied using the assay method described previously (14). The apparent K_m for CTP or P-Cho in the presence of different concentrations of the second substrate was determined. In a final volume of 50 µl, the LicC assay contained the substrates, CTP and phospho[methyl-¹⁴C]choline (specific activity 3.68 mCi/mmol), at the appropriate concentrations for each study indicated in the figure legends, and 10 mM MgCl₂ in 150 mM bis-Tris-HCl, pH 7.0. LicC was added last to start the reaction. After incubation of the reaction mixture at 37 °C for 10 min, the reaction was stopped by the addition of 5 µl of 0.5 M EDTA. A 40-µl aliquot of the reaction mixture was applied to a preabsorbent Silica Gel G plate, which was developed with 95% ethanol/2% ammonium hydroxide (1:1, v/v). The single product was visualized with a Bioscan Imaging detector and migrated with the standard CDP-Cho. Furthermore, different concentrations of either product (CDP-Cho and PP_i) of the LicC reaction were included in the assay to determine the order of the reaction.

Crystallization-- Se-Met LicC was concentrated to 23.3 mg/ml in 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA. The preliminary crystals of Se-Met LicC were obtained from Hampton Research screening kits, and the refined condition contained 0.1 M sodium citrate, 1.85 M ammonium sulfate, 0.2 M sodium/potassium tartrate, pH 5.8, at 18 °C. Crystals appeared after several days, which grew further for 1 week. These crystals were transferred to a cryoprotectant solution that was made by adding 30% glycerol to the mother liquor for data collection. Co-crystals of LicC and CDP-Cho were grown by adding CDP-Cho and MgCl₂ to the protein to a final concentration of 10 mM each, and the mixture was incubated overnight at 4 °C. The crystallization condition contained 0.4 M sodium acetate, 21% polyethylene glycol 4000, 0.1 M sodium cacodylate, pH 6.5, at 18 °C. The rectangular crystals grew in 10 days. A cryoprotectant solution for cryodata collection was made by adding 22.5% glycerol to the mother liquor.

Data Collection and Structure Determination-- Multiwavelength anomalous dispersion data were measured from a single crystal of Se-Met apo-LicC at beam line X8-C at the National Synchrotron Light Source, equipped with an ADSC Quantum 4R detector operating at 100 K. Two wavelengths near the selenium absorption edge, 0.9793 Å (the peak) and 0.9795 Å (the inflection point), were chosen by measuring the x-ray absorption spectrum of the protein crystal. The data sets from both wavelengths were collected, reduced, and scaled by the program HKL 2000 (24). The crystals of apo-LicC belong to space group P2₁ 2₁ 2₁ with unit cell dimensions of a = 42.6 Å, b = 63.6 Å, c = 122.4 Å. The program SOLVE was used to scale the two wavelength data sets together, to locate selenium positions, and to calculate the protein phases (25). Two of the four selenium atoms in the LicC enzyme were confirmed. The program RESOLVE was used for reciprocal space solvent flattening (26). The statistics of data collection and phasing are summarized in Table I. The atomic model was built using the programs Warp (27) and Xtalview (28), followed by crystallographic refinement in the program XPLOR (29). The refinement steps included simulated annealing, conjugated gradient minimization, and individual B-factor refinement. The refinement statistics are shown in Table II.

Complete diffraction data from a single crystal of the LicC·CDP-Cho·Mg²⁺ complex were measured at beamline 19-BM at the Advanced Photon Source, equipped with a SBC-2 CCD x-ray detector operating at 100 K. The program HKL 2000 (24) was used to integrate and scale the data set. The crystals of the ternary complex belonged to space group P1 with unit cell dimensions of a = 48.2 Å, b = 69.0 Å, c = 81.6 Å,  = 93.4°,  = 92.8°,  = 97.1°. The data collection statistics are summarized in Table I. The structure of the CDP-choline-bound LicC was determined by the molecular replacement method using the refined apo-enzyme as a search model. The cross-rotation and translation functions and Patterson correlation refinement were calculated using the program XPLOR (29). Using the data between 15.0 and 4.0 Å, the rotation function, followed by Patterson correlation refinement, gave four outstanding solutions that correspond to four molecules of LicC. Because any point can be taken as an origin in space group P1, we rotated the search model according to the highest solution of rotation function and considered this rotated molecule as the first molecule. With fixing the first molecule, the orientations of the other three molecules were determined by applying the three independent noncrystallographic symmetry operations to the first molecule, and then their positions relative to the first molecule were found by searching the x, y, and z translations in the unit cell. After the model, including all four molecules, was subjected to rigid body refinement at the resolutions between 6.0 and 4.0 Å in the program XPLOR, the resulting R factor was high (40.6%), suggesting the conformational difference of the CDP-choline-bound structure from the search model of the apo-enzyme structure. After the bulk solvent contribution was made to the intensities of reflections at low resolution, the high and low resolution limits were extended to 20 and 2.4 Å, respectively. Crystallographic refinement including simulated annealing, conjugate gradient minimization, and individual B-factor refinement was then performed at the extended resolutions. After the crystallographic refinement, the electron density map was improved by the noncrystallographic symmetry averaging method using the program DM (30). The F_o  F_c difference map clearly showed the electron density of CDP-Cho·Mg²⁺ in all four molecules, verifying the correct molecular replacement solutions. Several iterations of model building and the crystallographic refinement gave the excellent refinement statistics (Table II). Noncrystallographic restraints were applied to the main chain of each molecule for simulated annealing refinement. The final structure contains four molecules of LicC (residues 4-231 in each of four subunits).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Overview of the LicC Structure-- The apo-LicC structure reveals a mixed seven-stranded -sheet flanked by three -helices on one side (1, 2, and 6) and another three -helices on the other (3, 4, and 5) (Fig. 1). The seven-stranded sheet has six parallel strands (strands 1, 2, 3, 4, 5, and 7), and strand 6 is antiparallel to others, representing the typical nucleotide-binding fold (31). Four additional strands (5a, 5b, 5c, and 5d) located between strands 5 and 6, which are perpendicular to the seven-stranded -sheet, and another four strands (1a and 1b, 4a, and 7a) are not part of the main -sheet. Comparison of the apo-LicC structure with known structures in the Protein Data Bank using the DALI server (32) revealed that the two best matches were with two members of the NTP transferase superfamily: P. aeruginosa RmlA (Protein Data Bank code 1g0r, r.m.s. deviation 2.6 Å, C atoms of 218 residues) and GlmU (Protein Data Bank code 1fwy, r.m.s. deviation 2.8 Å, C atoms of 214 residues). The structures of two additional enzymes that belong to the NTP transferase superfamily were also compared using the program O (33): S. enterica RmlA (Protein Data Bank code 1iin, r.m.s. deviation 1.9 Å, C- atoms of 171 residues) and E. coli methylerythritol-4-phosphate cytidylyltransferase (Protein Data Bank code 1i52, r.m.s. deviation 1.7 Å, C- atoms of 131 residues). The finding of these low r.m.s. deviation values indicates that LicC is structurally similar to the three members of the NTP transferase superfamily. The structural similarity along with the conservation of the N-terminal signature sequence supports the classification of LicC as a member of the NTP transferase superfamily (14). Among the three members of the NTP transferase superfamily homologous to LicC, GlmU is an uridyltransferase involved in the production of UDP-N-acetylglucosamine, an essential precursor for the cell wall and membrane biosynthesis in bacteria (18). RmlA is a thymidylyltransferase and the first enzyme in the biosynthesis of deoxy-TDP-L-rhamnose that is a component of the cell wall in many bacteria (20). E. coli methylerythritol-4-phosphate cytidylyltransferase is a cytidyltransferase involved in the mevalonate-independent pathway for isoprenoid biosynthesis (19).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Overall stereoview of the LicC structure. Helices are shown in blue, strands in green, and other secondary structural elements in yellow. The core seven -stranded sheets and flanking -helices on either sides are labeled as 1-7 and 1-6. Additional -strands that are not part of the core sheet are labeled by alphabetizing after the preceding -strand number. For example, the -strand inserted between 4 and 5 is labeled as 4a. Figs. 1, 3, and 4 were produced by the program RIBBONS (40).

The crystals of LicC·CDP-Cho·Mg²⁺ ternary complex contain four molecules in an asymetric unit, assembled to form two dimers. However, purified LicC behaves as a monomer (14), and the apo-LicC structure contains one molecule per asymmetric unit, arguing that the dimers arise from the crystallization condition for the CDP-Cho·Mg²⁺ complex. The four molecules in the asymmetric unit superimpose to one another with an r.m.s. deviation of between 0.64 and 0.75 Å, illustrating that they have nearly identical conformations. The hydrophilic nature of the dimer interface (~60% of interactions) is also consistent with the conclusion that the monomer is the biologically functional unit. Additionally, sequence analysis of the NPT transferase superfamily members reveals that LicC lacks the oligomerization domain at the C terminus. The structures of apo-LicC and the LicC·CDP-Cho·Mg²⁺ complex superimpose with an r.m.s. deviation of 1.51 Å, indicating that only small structural changes occur upon CDP-Cho·Mg²⁺ binding to LicC.

The LicC Active Site-- CDP-Cho·Mg²⁺ is bound in a deep pocket with negative electrostatic potential (Fig. 2A). Bound Mg²⁺ is located between the pyrophosphate group of CDP-Cho and the active site pocket, neutralizing the charge repulsion between them. The negatively charged residues lining the active site pocket of LicC include Asp¹⁰⁵, Asp¹⁰⁷, Glu¹³⁵, Asp¹⁹², Glu²¹⁶, and Asp²¹⁸. Among these residues, Asp¹⁰⁷ and Glu²¹⁶ are conserved within the NTP transferase family members except GlmU and E. coli methylerythritol-4-phosphate cytidylyltransferase. In GlmU, while Asp¹⁰⁷ is conserved, Glu²¹⁶ is changed to Gly, and in E. coli methylerythritol-4-phosphate cytidylyltransferase, Asp¹⁰⁷ and Glu²¹⁶ are replaced by Ala and Lys, respectively. Consistent with the conserved number of negatively charged residues, the active site electrostatic potential of LicC (Fig. 2A) and RmlA (Fig. 2B) are highly negative. The GlmU active site (Fig. 2C) is slightly less negatively charged compared with LicC and RmlA. In contrast, the methylerythritol-4-phosphate cytidylyltransferase active site (Fig. 2D) is positively charged.

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Solvent-accessible surface and electrostatic surface potential. Red and blue represent negative and positive electrostatic surface potential, respectively, whereas white represents neutral electrostatic surface potential. Bound ligands are shown with a ball and stick representations. A, the electrostatic potential of LicC with bound CDP-Cho·Mg2+. B, the electrostatic potential of RmlA from P. aeruginosa with bound dTDP-D-glucose (PDB code 1g1l). C, the electrostatic potential of GlmU with bound UDP-N-acetylglucosamine (PDB code 1fwy). D, the electrostatic potential of methylerythritol-4-phosphate cytidylyltransferase from E. coli with bound CTP (PDB code 1i52). We used the coordinates of the CTP-bound structure in D, because the product-bound structure is not available. This figure was produced with the program GRASP (41).

Interaction of the cytosine base of CDP-Cho with the enzyme explains the observed specificity of LicC for CTP (14). The O-2 oxygen of the cytosine base is hydrogen-bonded to the amide nitrogen of Ala¹³ and the hydroxyl group of Ser⁹⁰, and the N-4 nitrogen of the base is hydrogen-bonded to the carbonyl oxygens of Tyr⁸² and Tyr⁸⁵ (Fig. 3A). It is clear that the N-4 nitrogen interaction excludes binding of other pyrimidine bases (i.e. thymine and uracil) because their corresponding atom (i.e. O-4 oxygen) cannot form hydrogen bonds with the carbonyl oxygens of Tyr⁸² and Tyr⁸⁵. There is no specific interaction of the C-5 and C-6 carbons of the cytosine base to the protein. The pyrimidine ring is stacked between the loop of strands 1 and 1a and the loop of strand 3 and helix 3, suggesting that larger purine bases can, if not ideally, also fit between these two loops. However, only the adenine base, and not the guanine base, is allowed, because the N-6 nitrogen of the adenine base, but not the O-6 oxygen of the guanine base, can form hydrogen bonds with Tyr⁸² and Tyr⁸⁵. Finally, the hydrogen bond interactions of 2'- and 3'-hydroxyl groups of the ribose sugar with the amide nitrogens of Gly¹⁴ and Ala¹⁰⁶ and the carbonyl oxygen of Leu¹¹ secure the base of CTP, and potentially ATP, in the binding pocket.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The CDP-Cho binding pocket. A, interactions of the cytidine group of CDP-Cho with the enzyme. Note that hydrogen bond interactions of the N-4 nitrogen of the cytosine base with the carbonyl oxygens of Tyr82 and Tyr85 confer the nucleotide specificity of LicC for CTP. B, interactions of the diphosphocholine group of CDP-Cho and Mg2+ with the enzyme. Six oxygen atoms from the side chains of Asp107, Glu216, and Asp218, the pyrophosphate of CDP-Cho, and a water molecule are octahedrally arranged about Mg2+ (magenta). The side chain  electrons of Trp136, Tyr190, and Asp192 interact with the trimethylammonium group of CDP-Cho. This type of interaction is observed in other proteins that recognize the trimethylammonium group of choline (34-36). The closest distances between the side chains of three residues and the trimethylammonium groups are 3.5, 3.7, and 3.2 Å, respectively.

It is known that the trimethylammonium group of choline is recognized by electron-rich aromatic rings (34-36). Indeed, in the LicC·CDP-Cho·Mg²⁺ complex, the binding pocket of the trimethylammonium group is formed by the aromatic rings of Tyr¹⁹⁰ and Trp¹³⁶ and the carboxyl group of Asp¹⁹². The side chains compensate for the positive charge of the trimethyl ammonium group through a stabilizing interaction with their  electrons (Fig. 3B). We hypothesize that the negative charge of Asp¹⁹² may also be a factor in binding of the trimethylammonium group, because an electrostatic bond is stronger than a cation- bond (37).

Magnesium is required for the catalytic activity of LicC (14). In the LicC·CDP-Cho·Mg²⁺ structure, the magnesium atom is fixed in space by interactions with six oxygen atoms, two oxygens from each of the - and -phosphates of CDP-choline, the carboxyl groups of Asp¹⁰⁷, Glu²¹⁶, and Asp²¹⁸, and a water molecule, forming an octahedral geometry (Fig. 3B). This bound water molecule is further hydrogen-bonded to the carboxyl group of Asp¹⁰⁵. The -phosphate oxygen of CDP-Cho and the carboxyl groups of Asp¹⁰⁷ and Asp²¹⁸, which bind the magnesium, form hydrogen bonds with the amino group of Lys²⁸ in the signature sequence (Fig. 3B), suggesting that Lys²⁸ participates in orienting the side chains of Asp¹⁰⁷ and Asp²¹⁸ for binding both magnesium and the nucleotide substrate. The interaction between the amino group of Lys²⁸ and the -phosphate oxygen can enhance the catalytic reaction rate by stabilizing the developing negative charge in the pentacoordinate transition state. When the corresponding Lys of NTP transferases from other organisms was mutated, the catalytic activity was decreased (18, 19), which is consistent with the proposed role of Lys²⁸ of LicC in catalysis. A -phosphate oxygen that does not bind magnesium is hydrogen-bonded to the hydroxyl group of Tyr¹⁹⁰ that also interacts with the trimethylammonium group via its aromatic ring (Fig. 3B).

Structural Differences between LicC and LicC·CDP-Cho·Mg²⁺-- The most significant change in the secondary structural elements induced by CDP-Cho·Mg²⁺ binding involves the four strands (5a, 5b, 5c, and 5d) located between strands 5 and 6. In the LicC·CDP-Cho·Mg²⁺ structure, strands 5a and 5b with their intervening loop become the continuous, long strand 5a, and strands 5c and 5d with their intervening loop coalesce to form strand 5c (Fig. 4A). These new, longer strands 5a and 5c are interconnected by a hydrogen bond network to form a two-stranded  sheet.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Structural changes in the LicC·CDP-Cho·Mg2+ ternary complex. A, when CDP-Cho·Mg2+ binds to the apo-LicC (cyan), conformational changes in four strands (5a, 5b, 5c, and 5d) occur, resulting in the formation of two strands (5a and 5c, shown in red). First two strands (5a and 5b) are merged to be a new long strand 5a and the last two strands (5c and 5d) to be a new long strand, 5c. Additional conformational change occurs in 7a and its C-terminal loop (red). The secondary structural elements of the apo-LicC structure are labeled as in Fig. 1, and the new long strands 5a and 5c of the LicC·CDP-Cho·Mg2+ ternary complex (red) are labeled and highlighted in an open rectangular box. Carbons of CDP-Cho are shown in yellow, and Mg2+ is shown in magenta (ball and stick representations). B, the side chain conformations of several residues (gray in apo-LicC and blue in the LicC·CDP-Cho·Mg2+ ternary complex) affected by binding of CDP-Cho·Mg2+ are shown in ball and stick representations. The salt bridge between Arg129 and Glu216 in apo-LicC is broken in the LicC·CDP-Cho·Mg2+ ternary complex due to the movements of Glu216 along with Asp218 for interacting with Mg2+, which are illustrated by arrows. Note that upon binding of CDP-Cho·Mg2+, the side chains of Trp136 and Tyr190 move inward to optimize the fit for the trimethylammonium group of Cho as it is shown in Fig. 3. C, Arg18 in apo-LicC is anchored to the bottom of the active site by forming hydrogen bonds with the side chain of Asp107 and the carbonyl oxygen of Ile217, and in the LicC·CDP-Cho·Mg2+ ternary complex, the same residue swings out and is exposed to solvent.

CDP-Cho·Mg²⁺ binding also alters the side chain positions of several residues. Side chains of Glu²¹⁶ and Asp²¹⁸ move toward bound magnesium by 7.4 and 3.8 Å, respectively, to coordinate the magnesium (Fig. 4B). Since Glu²¹⁶ in the apoenzyme structure is salt-bridged to Arg¹²⁹, the movement of Glu²¹⁶ disrupts the salt bridge interaction with Arg¹²⁹, displacing the guanidinium group of Arg¹²⁹, breaking the hydrogen bond interaction of Arg¹²⁹ with the amide nitrogen of Gly²¹¹. The displaced guanidinium group of Arg¹²⁹ is hydrogen-bonded to the side chain of Glu¹³⁵. Also, the side chains of Trp¹³⁶ and Tyr¹⁹⁰ move toward the trimethylammonium group of CDP-choline. Arg¹²⁹, Glu¹³⁵, and Trp¹³⁶ are all located in the newly formed strand 5a.

Arg¹⁸ (LicC numbering) in the signature sequence of NTP transferase structures interacts with the nucleotide triphosphate (19-21). In the apo-LicC structure, the guanidinium group of Arg¹⁸ points down to the active site pocket, forming a hydrogen bond network with the carboxyl group of Asp¹⁰⁷ and the carbonyl oxygen of Ile²¹⁷ (Fig. 4C). In contrast, the guanidinium group is flipped out and exposed to solvent in the LicC·CDP-Cho·Mg²⁺ complex (Fig. 4C). Since the LicC·CDP-Cho·Mg²⁺ complex is the structure of the enzyme intermediate immediately after the release of PP_i (see below), the solvent-exposed guanidinium group of Arg¹⁸ may correspond to the conformational change that facilitates the release of PP_i to the solvent. The side chain of the corresponding Arg in E. coli methylerythritol-4-phosphate cytidylyltransferase forms hydrogen bonds with the - and -phosphate oxygens of CTP (19). In LicC, after adjusting the torsion angles of Arg¹⁸, similar interactions can be formed between the side chain of Arg¹⁸ and the - and -phosphate oxygens of CTP modeled using the CTP coordinates of E. coli methylerythritol-4-phosphate cytidylyltransferase (Protein Data Bank code 1i52). This finding suggests that Arg¹⁸ may be involved in the release of PP_i and offset the negative charge developed on the -phosphate oxygen of CTP during the catalytic reaction.

Kinetic Mechanism for LicC-- A series of kinetic experiments were performed to define the kinetic mechanism for LicC. Double reciprocal plots of 1/v versus 1/[CTP] at different concentrations of P-Cho (Fig. 5A) intersected far to the left of the 1/v axis and far below the 1/[CTP] axis. The same pattern is observed in case of 1/v versus 1/[P-Cho] in the presence of different concentrations of CTP (Fig. 5B). A cursory inspection of the graphs leaves the impression that the lines are parallel, which would indicate a ping-pong mechanism. However, they are not, and there are many cases of sequential enzymes where lines appear to be parallel. In these cases, the enzyme has a relatively high affinity for the leading substrate, but because of the relative values of the other rate constants the apparent K_m value is much larger than the equilibrium binding constant (38). Product inhibition studies were performed to verify that LicC is a sequential enzyme and to establish the order of addition of substrates. CDP-Cho is a competitive inhibitor with respect to CTP, whereas PP_i is a mixed-type inhibitor with respect to CTP (Fig. 6). These data show that CTP and CDP-Cho bind to the same form of the enzyme. On the other hand, both PP_i and CDP-Cho are mixed-type inhibitors with respect to P-Cho (Fig. 7). This pattern of the product inhibition is characteristic of a sequential bi bi reaction with CTP as the leading substrate and CDP-Cho as the last product to leave, which is the same as the catalytic mechanism of E. coli glucose-1-phosphate thymidylyltransferase, a homologue of P. aeruginosa RmlA (39).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   LicC reaction kinetics mechanism. A, the double reciprocal plots of 1/v versus 1/[CTP] at different concentrations of P-Cho. B, the double reciprocal plots of 1/v versus 1/[P-Cho] at different concentrations of CTP. Both groups of the apparently parallel double reciprocal plots intersected far to the left of the 1/v axis and far below the 1/[CTP] or 1/[P-Cho] axis, characteristic for an ordered bi bi mechanism. Kinetic data were obtained using the assay described under "Experimental Procedures," and the error bars present the range of data.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Product inhibition of LicC reaction with varied CTP. A, double reciprocal plots of 1/v versus 1/[CTP] in the presence of different concentrations of CDP-Cho revealed that CDP-Cho is a competitive inhibitor for CTP. B, double reciprocal plots of 1/v versus 1/[CTP] in the presence of different concentrations of PPi revealed that PPi is a mixed-type inhibitor for CTP. The concentration of P-Cho (1 mM) was held constant in the experiments, which were performed using the assay described under "Experimental Procedures." Error bars indicate the range of data.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Product inhibition of the LicC reaction with respect to P-Cho. A, double reciprocal plots of 1/v versus 1/[P-Cho] in the presence of different concentrations of CDP-Cho revealed that CDP-Cho is a mixed-type inhibitor with respect to P-Cho. B, double reciprocal plots of 1/v versus 1/[P-Cho] in the presence of different concentrations of PPi revealed that PPi is a mixed-type inhibitor with respect to P-Cho. The concentration of CTP (160 µM) was held constant in the experiments. Kinetic data were obtained using the assay described under "Experimental Procedures," and the error bars present the range of data.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We propose a model for the catalytic cycle of LicC that is consistent with the kinetic and structural data (Fig. 8). CTP·Mg²⁺ is the leading substrate. The LicC nucleotide specificity for CTP is conferred by specific interactions that include the N-4 nitrogen of the cytosine base and the carbonyl oxygens of Tyr⁸² and Tyr⁸⁵, the CTP O₂ oxygen with the amide nitrogen of Ala¹³ and the hydroxyl group of Ser⁹⁰. The ,-phosphate oxygens are proposed to form a salt bridge with Arg¹⁸, which points into the active site of LicC. The planar aromatic ring of CTP is sandwiched between the loop of strands 1 and 1a and the loop of strand 3 and helix 3. The side chains of Asp¹⁰⁷, Glu²¹⁶, Asp²¹⁸, and the -phosphate of CTP coordinate the Mg²⁺. Movement of Glu²¹⁶ breaks the salt bridge with Arg¹²⁹ that in turn forms hydrogen bond to Glu¹³⁵. This induces four -strands in LicC into coalesce into two long -strands. This rearrangement causes the repositioning of Trp¹³⁶ and Tyr¹⁹⁰ to optimize the binding of the second substrate P-Cho and position the bound P-Cho for the CMP transfer reaction. The structure is consistent with the reaction occurring by in-line nucleophilic attack of the phosphate oxygen of P-Cho on the -phosphate of CTP, similar to other members of the NTP transferase superfamily. The first product released from the active site is PP_i, which may be facilitated by its interaction with Arg¹⁸, which points away from the active site in the product structure. CDP-Cho·Mg²⁺ is the final product released from the enzyme, allowing the Glu²¹⁶·Arg¹²⁹ salt bridge to reform and regeneration of the apo-enzyme structure.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   The LicC catalytic cycle. LicC operates by a sequential ordered mechanism that is interpreted based on the enzyme kinetics and the conformational differences between the structure of apo-LicC and the LicC·CDP-Cho·Mg2+ ternary complex. LicC first binds CTP·Mg2+, which triggers a conformation change that breaks the salt bridge between Glu216 and Arg129. This action reorganizes the -sheet structure of the protein to allow Glu216 to directly interact with the incoming Mg2+ (see "Discussion"). The subsequent binding of P-Cho is facilitated by the movement of Trp136 and Tyr190 to optimize the interaction of the enzyme with the incoming trimethylammonium group of P-Cho. Catalysis occurs in the ternary complex, and PPi is released first, facilitated by the movement of Arg18. CDP-Cho·Mg2+ is the last product released from the enzyme, which allows the Glu216·Arg129 salt bridge to reform and regenerate the apo-enzyme structure.

The structural changes in the LicC·CDP-Cho·Mg²⁺ ternary complex are confined to the residues and secondary structural elements that create the binding sites for Mg²⁺ and the trimethylammonium group of Cho. These important conformational changes initiate at the Mg²⁺ binding site and are propagated to the binding site of the trimethylammonium group via four strands (5a, 5b, 5c, and 5d). LicC binds Mg²⁺ by breaking the salt bridge between Glu²¹⁶ and Arg¹²⁹ in the apo-enzyme to release Glu²¹⁶ to coordinate the Mg²⁺. The side chain of Glu¹³⁵ forms a hydrogen bond with the side chain of Arg¹²⁹, compensating for the absence of Glu²¹⁶. Breaking the salt bridge triggers the rearrangement of four individual strands (5a, 5b, 5c, and 5d) into two longer strands (5a and 5c) (Fig. 4A). This reorganization displaces the side chains of Trp¹³⁶ on strand 5a and Tyr¹⁹⁰ on the loop of helices 4 and 5 to position P-Cho for an in-line nucleophilic attack of the phosphate oxygen of P-Cho on -phosphorus of CTP. Phosphoethanolamine lacks the trimethylammonium group of P-Cho and therefore can fit in the active site. However, the positioning of phosphoethanolamine within the active site pocket for the CMP transfer reaction is predicted to be less efficient than P-Cho, accounting for the lower catalytic activity of LicC when phosphoethanolamine is the substrate (14).

Our structures expand the understanding of the catalytic mechanism of members of the NTP transferase superfamily by implicating Mg²⁺ as an integral component in catalysis. The Mg²⁺ in previously determined NTP transferase structures is postulated to stabilize the PP_i leaving group, and thus plays an indirect role in catalysis (19, 21). However, the Mg²⁺ bound at the active site of the LicC·CDP-Cho·Mg²⁺ structure suggests the direct involvement of the Mg²⁺ in the catalytic mechanism of LicC. We propose two direct roles for Mg²⁺ in NTP transferases. First, the bound Mg²⁺ accelerates catalysis by positioning the phosphate oxygen of P-Cho adjacent to the -phosphate of CTP to align the incoming second substrate of in-line nucleophilic attack. Second, the interactions of the positively charged Mg²⁺ works in concert with the side chains of Arg¹⁸ and Lys²⁸ to stabilize the developing negative charge in the pentacoordinate transition state during CMP transfer.

The role of Arg¹⁸ and Lys²⁸ in the in-line nucleophilic substitution mechanism proposed for LicC is the same as proposed for the structurally known members of the NTP transferase family, GlmU, RmlA, and E. coli methylerythritol-4-phosphate cytidylyltransferase. When the nucleotide and phosphosugar substrates bind to GlmU to form the ternary complex, the proximity of the substrates and the catalytic Arg¹⁸ (also Arg¹⁸ in LicC) aids the reaction by stabilizing a charged transition state. In RmlA, three residues, Arg¹⁵, Lys¹⁶², and Arg¹⁹⁴, appear important in the catalytic mechanism. Arg¹⁵ (Arg¹⁸ in LicC) also functions by stabilizing the charged transition state, and Lys¹⁶² (not conserved in LicC) interacts with the phosphate of the phosphosugar substrate to increase its nucleophilicity. The function of Arg¹⁹⁴ (not conserved in LicC) is less clear because the side chain conformation is different in two RmlA structures such that the side chain of Arg¹⁹⁴ in S. enterica RmlA interacts with the phosphate of the phosphosugar substrate, whereas its side chain in P. aeruginosa RmlA does not. In E. coli methylerythritol-4-phosphate cytidylyltransferase, Arg²⁰ and Lys²⁷ (Arg¹⁸ and Lys²⁸ in LicC, respectively) interact with the -phosphate of CTP and are considered to function in transition state stabilization. Arg¹⁵⁷ from the second subunit of the dimer (not conserved in LicC) and Lys²¹³ (Glu²¹⁶ in LicC that coordinates Mg²⁺) interact with the phosphate of the phosphoalcohol substrate, increasing the nucleophilicity of the phosphoalcohol during the catalytic reaction. Taken together, these results suggest that Arg¹⁸ and Lys²⁸ (LicC numbering) in the signature sequence play a common role in transition state stabilization and substrate activation in catalytic mechanism for the NTP transferase family members.

The active sites of GlmU and RmlA have negative electrostatic potential that is unfavorable for binding of the negatively charged substrates. Mg²⁺-coordinating Asp¹⁰⁷ and Glu²¹⁶ in LicC correspond to Asp¹¹⁰ and Asp²²⁵ in RmlA and Asp¹⁰⁵ and Gly²²⁵ in GlmU. In RmlA, these conserved Asp residues contribute a putative, LicC-like metal binding site, but metal binding was not observed in the structures of RmlA (19, 21). Mg²⁺ may bind to Asp¹¹⁰ and Asp²²⁵ of RmlA to function as catalytic metal. The side chain of Asp¹¹⁰ in P. aeruginosa RmlA is within hydrogen bond distance to the -phosphate oxygen of the nucleotide substrate, and the charge repulsion between Asp¹¹⁰ and the -phosphate of the nucleotide substrate must be compensated during the catalytic reaction. In LicC, Mg²⁺ bound by Asp¹⁰⁷ and Glu²¹⁶ neutralizes the negative charges of the substrates and increasing the catalytic activity by stabilizing the transition state. Interestingly, Mg²⁺-coordinating Asp¹⁰⁷ and Glu²¹⁶ in LicC are replaced by Ala¹⁰⁸ and Lys²¹³ in E. coli methylerythritol-4-phosphate cytidylyltransferase, respectively. These two replacements not only explain why the active site of E. coli methylerythritol-4-phosphate cytidylyltransferase has positive electrostatic potential but also suggest that this enzyme may not require the direct involvement of Mg²⁺ for binding substrates and catalysis.

	ACKNOWLEDGEMENTS

We thank the Protein Production Facility for protein purification and preliminary crystallization trials. We also thank Matthew Frank for technical assistance and Drs. Li-Wei Hung at (National Synchrotron Light Source) and Rongguang Zhang (Advanced Photon Source) for help with diffraction data collection.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM 45737 (to S. J.) and GM 34496 (to C. O. R.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.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 1jyk and 1jyl) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

To whom correspondence should be addressed. Tel.: 901-495-3838; Fax: 901-495-3032; E-mail: hee-won.park@stjude.org.

Published, JBC Papers in Press, November 12, 2001, DOI 10.1074/jbc.M109163200

	ABBREVIATIONS

The abbreviations used are: P-Cho, phosphocholine; CDP-Cho, cytidyldiphosphocholine; CCT, CTP:phosphocholine cytidylyltransferase; r.m.s., root mean square.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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