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J. Biol. Chem., Vol. 278, Issue 32, 30022-30027, August 8, 2003

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Crystal Structure of 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, an Enzyme in the Non-mevalonate Pathway of Isoprenoid Synthesis^*

Takashi Wada   ¶, Tomohisa Kuzuyama ||, Shinya Satoh , Seiki Kuramitsu , Shigeyuki Yokoyama   **, Satoru Unzai ¶, Jeremy R.H. Tame ¶ and Sam-Yong Park ¶ 

From the Genomic Sciences Center, RIKEN Yokohama Institute,1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, ^||Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-0032, RIKEN Harima Institute, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, and ^¶Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, ^**Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

Received for publication, April 25, 2003 , and in revised form, May 22, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The crystal structure of the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) kinase from the thermophilic bacterium Thermus thermophilus HB8 has been determined at 1.7-Å resolution. This enzyme catalyzes phosphorylation of the 2-hydroxyl group of CDP-ME, the fourth step of the non-mevalonate pathway, which is essential for isoprenoid biosynthesis in several pathogenic microorganisms. Since this pathway is absent in humans, it is an important target for the development of novel antimicrobial compounds. The structure of the enzyme is similar to the structures of mevalonate kinase and homoserine kinase, members of the GHMP superfamily. Lys⁸ and Asp¹²⁵ are active site residues in mevalonate kinase that also appear to play a catalytic role in CDP-ME kinase. Both the mevalonate and the non-mevalonate pathways therefore involve closely related kinases with similar mechanisms. Assaying the enzyme showed that CDP-ME kinase will phosphorylate CDP-ME but not 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol, indicating the substrate pyrimidine moiety is involved in important interactions with the enzyme.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Isoprenoids are a hugely diverse group of molecules found in all organisms and are constructed from just two building blocks: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Most eukaryotes use the mevalonate pathway to synthesize IPP from acetyl CoA via mevalonate (1), but many bacteria and plant plastids use the "non-mevalonate" pathway to produce IPP and DMAPP (2). This pathway starts with pyruvate and glyceraldehyde-3-phosphate (3) and involves seven enzymes (Fig. 1). Both the sixth and seventh steps require dioxygen-free conditions (4, 5). At the final step, IPP and DMAPP are produced in a ratio of 5:1 (5, 6). Since this pathway is not found in mammals but is found in several pathogenic microorganisms, for example, Mycobacterium tuberculosis and the malaria parasite Plasmodium falciparum, it is an important target for development of novel antimicrobial compounds. The antibiotic fosmidomycin inhibits the second step in the non-mevalonate pathway (7), and it is able to cure mice of Plasmodium vinckei (8). The fourth step of the non-mevalonate pathway involves the phosphorylation of 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol (CDP-ME). The enzyme CDP-ME kinase (CMK, EC 2.7.1.148 [EC] ) converts CDP-ME into CDP-ME 2-phosphate (CDP-ME2P) in an ATP-dependent reaction (9–11). This enzyme belongs to the GHMP kinase superfamily (12–14) named after galactokinase (EC 2.7.1.6 [EC] ), homoserine kinase (HSK, EC 2.7.1.39 [EC] ), mevalonate kinase (MVK, EC 2.7.1.36 [EC] ), and phosphomevalonate kinase (EC 2.7.4.2 [EC] ). Three members of this superfamily, MVK, phosphomevalonate kinase, and mevalonate-5-diphosphate decarboxylase (EC 4.1.1.33 [EC] ), catalyze the third to fifth reactions in the mevalonate pathway (1). HSK catalyzes the formation of O-phospho-L-homoserine from L-homoserine in the threonine biosynthesis pathway. Three conserved motifs have been identified in the GHMP superfamily (12, 15). These motifs are involved in the formation of the active site (16). We report here the crystal structure of CMK from an extremely thermophilic bacterium, Thermus thermophilus HB8, and compare it with those of other enzymes in the GHMP superfamily. The sequence of T. thermophilus HB8 CMK consists of 275 amino acid residues and is highly similar to its homologues from Escherichia coli and M. tuberculosis, with 33 and 35% sequence identity, respectively (Fig. 2). Additionally, we demonstrate the substrate specificity of CMK. Several residues in the enzyme are identified that may be important for the enzyme activity, based on comparison with homologous structures.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Non-mevalonate pathway. The pathway is composed of seven reaction steps and starts with pyruvate and glyceraldehyde-3-phosphate. The enzyme CDP-ME kinase converts CDP-ME into CDP-ME 2-phosphate at the fourth step of the pathway. DXP, 1-deoxy-D-xylulose 5-phosphate; MECDP, 2-C-methyl-D-erythritol 2,4-cyclo-diphosphate; HMBDP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate.

View larger version (68K): [in this window] [in a new window]   FIG. 2. Structure-based sequence alignments of the enzymes of the GHMP kinase superfamily. The crystal structures of MVK from R. norvegicus and HSK from M. jannaschii were fitted onto that of CMK from T. thermophilus HB8 by DALI (29). The sequences of CMK from E. coli and M. tuberculosis are also aligned with the T. thermophilus HB8 sequence. Regions in the other enzymes equivalent to the CMK structure are underlined. The -helices and -strands are indicated as green and red letters, respectively. The secondary structures at CMK are also represented by green bars for -helices and red arrows for -strands. The boundary between the two domains is shown by a vertical magenta line. The three conserved motifs of the GHMP superfamily are indicated by blue bars. Numbers of the residues omitted from the alignment are indicated in square brackets. The residues shown in bold are identical in the three aligned CMK sequences. The residue numbers are given in a column on the right side of the sequence alignments. The GenBankTM accession numbers for these protein sequences are indicated at end of the alignments. The cited sequence of M. jannaschii HSK is based on the GI number 14195670.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Protein Preparation—E. coli B834(DE3) cells were transformed with pET11a carrying the T. thermophilus HB8 CMK gene (ychB) and then grown with shaking for 21 h at 37 °C in LeMaster medium with 10 g/liter lactose and 25 mg/liter seleno-L-methionine (17, 18). The cells were harvested and suspended in lysis buffer (20 mM Tris-HCl (pH 8.0) and 50 mM NaCl). The cell suspension was sonicated and incubated at 70 °C for 10 min. After centrifugation, ammonium sulfate was added to the recovered supernatant up to 600 mM final concentration. The protein was applied to a Resource PHE column (Amersham Biosciences) with initial buffer (50 mM sodium phosphate (pH 7.0) and 600 mM ammonium sulfate) and eluted with a linear gradient of elution buffer (50 mM sodium phosphate (pH 7.0)). The fractions containing CMK were pooled. Ammonium sulfate was added to the solution up to 2.1 M final concentration and centrifuged. The pellet was resuspended in 20 mM Tris-HCl (pH 8.0) and then applied to a HiPrep desalting column (Amersham Biosciences) washed with the same buffer. The desalted solution was applied to a HiTrap Heparin column (Amersham Biosciences) and eluted with 20 mM Tris-HCl (pH 8.0) and 400 mM NaCl. The target fractions were collected and applied to a Superdex 75 gel filtration column (Amersham Biosciences) with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The pooled CMK was applied to a HiPrep desalting column with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The protein solution was concentrated up to 2.2 mg/ml by centrifugation with a Vivaspin filter with 10-kDa molecular mass cutoff (Vivascience) and stored at 4 °C. The yield was 4.4 mg from 28 g of wet cells.

Crystallization and Structure Determination—Crystals of CMK were grown at 20 °C in a hanging drop consisting of 2 µl of the protein solution and 1 µl of mother liquor. The mother liquor consisted of 33 mM Tris-HCl (pH 8.5), 67 mM sodium acetate, 13% isopropanol, 8% butanol, and 13% polyethylene glycol 4000. The crystals belong to the space group P2₁2₁2₁, with cell dimensions a = 48.6 Å, b = 66.2 Å, c = 76.4 Å, and contain one molecule/asymmetric unit.

The structure of CMK was solved by multiple-wavelength anomalous dispersion. The crystal was cryo-cooled to –173 °C in the mother liquor with 30% glycerol. Diffraction data were collected with a CCD detector (Mar) at BL44B2, SPring-8, Harima, Japan (19). The data were integrated and scaled with HKL2000 and SCALEPACK (20). The positions of selenium atoms were determined by the program SOLVE (21) and followed by density modification with the program RESOLVE (22). The model was built with the program TURBO-FRODO (23). Structural refinement was carried out using the program REFMAC in the CCP4 program suite (24). The data statistics are given in Table I. The secondary structure was defined using the program PROCHECK (25).

Assay for CMK—Radioactive CDP-ME, UDP-ME, and ADP-ME were synthesized using 20 µl of the reaction mixtures, 50 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 2.5 mM dithiothreitol, 2.5 mM 2C-methyl-D-erythritol-4-phosphate (MEP) (26, 27), 0.25 mM [2-¹⁴C]MEP (851 MBq/mmol), 50 mg/liter E. coli MEP cytidylyltransferase (28), and 2.5 mM CTP, UTP, and ATP, respectively. After incubation at 37 °C for 1 h, the reactions were terminated by heat treatment at 70 °C for 10 min. 1 µg of T. thermophilus HB8 and E. coli CMK (10) and 0.5 µl of 100 mM ATP were then added to these mixtures. After incubation for 1 h at 50 °C for T. thermophilus HB8 CMK and 37 °C for E. coli CMK, 0.5 µl of these mixtures were applied to thin-layer chromatography on a cellulose gel (Merck). The cellulose plate was developed with a mixture of 2-butanol, acetic acid, and water (3:2:2) and then exposed to an imaging plate (Fujifilm) in which the intensity of photostimulated luminescence was proportional to the adsorbed radiation energy. The final autoradiogram was produced with a BAS-1500 image reader (Fujifilm).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Overall Structure of CMK—The structure of T. thermophilus HB8 CMK was refined to 1.7-Å resolution (Fig. 3a). The residues Met¹ to Gly²⁶⁸ were observed clearly in the final 2F_o – F_c electron density map (Fig. 4a), but the C-terminal region from Asp²⁶⁹ to Ala²⁷⁵ was apparently disordered and not visible in the map. The monomer structure consists of two domains (Figs. 2 and 3b). The N-terminal domain from Met¹ to Ala¹⁴⁸ consists of four -helices (H1–4) and six -strands (S1–6). The C-terminal domain from Leu¹⁴⁹ to Ala²⁷⁵ consists of five -helices (H5–9) and four -strands (S7–10). DALI searches (29) indicated that the structure of CMK is extremely similar to those of other enzymes in the GHMP superfamily; Methanococcus jannaschii MVK (Protein Data Bank code 1kkh [PDB] , z-score = 20.8) (16), HSK (PDB code 1fwl [PDB] , 20.3) (15), Rattus norvegicus MVK (PDB code 1kvk [PDB] , 18.5) (30), phosphomevalonate kinase (PDB code 1k47 [PDB] , 15.4) (31), and mevalonate-5-diphosphate decarboxylase (PDB code 1fi4 [PDB] , 13.2) (14). The structure-based sequence alignment by DALI indicates that the secondary structure of CMK is extremely similar to all these enzymes (Fig. 2). Z-scores were below 4.0 for other proteins in the Protein Data Bank. The three known conserved motifs of the GHMP superfamily are found in CMK at identical positions of those of HSK (15): motif 1 from Val⁹ to Ser¹⁴, motif 2 from Pro⁸⁶ to Ser⁹⁶, and motif 3 from Met²³⁰ to Ala²³⁶ (Fig. 2).

View larger version (43K): [in this window] [in a new window]   FIG. 3. Overall structure of CMK. a, a ribbon diagram of CMK showing the secondary structure. -helices and -strands are shown in green and red, respectively. b, a stereo view of the structure of CMK in the same orientation as in panel a. The N-terminal and C-terminal domains are shown in cyan and green, respectively. The conserved motifs 1, 2, and 3 (m1–3) are indicated in red. The amino acid residues Gly123, Asp125, and Thr165 may be important for the enzyme activity and are indicated in magenta. ATP-magnesium from R. norvegicus MVK (blue) and ADP-magnesium from M. jannaschii HSK (yellow) are superimposed on the structure of T. thermophilus HB8 CMK. CDP-ME (salmon) was docked by eye in the putative active site to show that the S1–S2 loop may interact with the substrate pyrimidine, although it is relatively far from the possible catalytic residues.

View larger version (69K): [in this window] [in a new window]   FIG. 4. Putative catalytic site of CMK. a, a stereo 2Fo – Fc electron density map with a ball-and-stick model around Lys8, Phe30, and Asp125, which are represented at 1.3 contour level. b, ADP (blue) and ATP (yellow) modeled in anti and syn conformations, respectively. The side chains of Lys8, Asn58, Lys83, Ser95, and Asp125 are represented as ball-and-stick models, and oxygen atoms and nitrogen atoms in these side chains are colored red and blue. The orientation is rotated about 90° around the vertical axis and turned 90° clockwise from that of Fig. 3.

The crystallographic model of T. thermophilus HB8 CMK shows no evidence of self-association of the protein. Since there is a single molecule in the asymmetric unit, the only symmetry elements present in the crystal are the 2₁ screw axes. In contrast, other members of the GHMP superfamily mostly form dimers. HSK dimerizes through hydrophobic helices in the C-terminal domain and strands in the N-terminal domain (15). Mammalian MVKs dimerize in solution, although M. jannaschii MVK apparently does not (16). R. norvegicus MVK forms a similar dimer to HSK, but more elongated due to different orientation of the two monomers (30). The dimer interface and relative orientation of the partner chains do not appear to be conserved among GHMP superfamily dimers, and dimerization plays no apparent functional role in these proteins.

Putative ATP and Magnesium-binding Site—The complex structures of R. norvegicus MVK with ATP-magnesium (PDB code 1kvk [PDB] ) and M. jannaschii HSK with ADP-magnesium (PDB code 1fwk [PDB] ) showed that motif 2 forms a phosphate-binding loop. ATP bound to MVK is an anti conformation (30), but ADP adopts a rare syn conformation when complexed to HSK (15) (Figs. 3b and 4b). The amino acid residues bonding to ATP in R. norvegicus MVK are Lys¹³, Asn⁵⁵, Asn¹⁰⁴, Ser¹⁰⁸, Ser¹³⁵, and Gly¹⁴² (30), and those in HSK are Asn⁵⁸, Val⁵⁹, Lys⁸³, Ser⁹⁷, and Thr¹⁷⁹ (15, 32). Thr¹⁷⁹ in HSK is equivalent to Thr²⁴³ in R. norvegicus MVK; mutation of this residue decreases the enzyme activity (33). Apart from this threonine, all the ATP-binding residues are in the N-terminal domain. Overlapping R. norvegicus MVK and M. jannaschii HSK onto T. thermophilus HB8 CMK shows that Asn⁵⁸ and Lys⁸³ in T. thermophilus HB8 CMK locate near ATP or ADP (Fig. 4b), and these residues may be important for ATP binding.

A magnesium ion is found bound to Ser¹⁴⁶ and Glu¹⁹³ in R. norvegicus MVK (30) and to Glu¹²⁶ in HSK (32), in both cases binding to the - and -phosphate groups of ATP. Mutations at Ser¹⁴⁶ in human MVK (identical to Ser¹⁴⁶ in R. norvegicus MVK) and Glu¹⁹³ in R. norvegicus MVK cause a decrease in enzyme activity (33, 34). However, these residues are not conserved in T. thermophilus HB8 CMK. Ser¹⁴⁶ in R. norvegicus MVK is equivalent to Gly⁹⁴ in T. thermophilus HB8 CMK, and both Glu¹⁹³ in R. norvegicus MVK and Glu¹²⁶ in M. jannaschii HSK are equivalent to Gly¹²³ in T. thermophilus HB8 CMK (Fig. 2). The crystal structure suggests that Ser⁹⁵ and Asp¹²⁵ might provide alternative ligands to hold a magnesium ion (Figs. 3b and 4b). Ser⁹⁵ neighbors Gly⁹⁴, which is equivalent to Ser¹⁴⁶ in R. norvegicus MVK. Superimposing the magnesium ion in R. norvegicus MVK onto T. thermophilus HB8 CMK shows that the metal ion center would be 1.4 Å from the -oxygen atom of Ser⁹⁵ and 3.3 Å from the side-chain carboxyl group of Asp¹²⁵. The position corresponding to the magnesium ion in M. jannaschii HSK, however, is further from these side chains, 4.9 Å from Ser⁹⁵ and 5.8 Å from Asp¹²⁵.

Catalytic Mechanism—From the structure of MVK bound to ATP, Fu et al. (30) suggested that the catalytic mechanism of the enzyme involves Asp²⁰⁴ acting as a catalytic base, removing a proton from the acceptor (C5) hydroxyl of mevalonate. In MVK, the carboxyl group of the side chain of Asp²⁰⁴ makes a salt bridge with the amine group on the side chain of Lys¹³, which in turn makes a close interaction with the -phosphate group of ATP. The C5 hydroxyl sits about 4 Å from the aspartate and the -phosphate. The amine group of Lys¹³ also interacts with the C5 hydroxyl group of mevalonate and lowers its pK_a for efficient catalysis. This role for the aspartate has several precedents including phosphofructokinase (35) and hexokinase (36), and mutation of this residue to asparagine reduced activity by a factor of about 10⁴ (34). Kinetic studies also showed that mutating Lys¹³ to methionine in R. norvegicus MVK causes about 60-fold diminution in the V_max value (37). Structural comparison of MVK and HSK, however, shows that Lys¹³ and Asp²⁰⁴ of MVK correspond to Thr¹⁰ and Asn¹³⁷ in M. jannaschii HSK, respectively, and the catalytic mechanism of MVK cannot be valid for HSK (30).

The catalytic lysine and aspartate residues of MVK correspond to Lys⁸ and Asp¹²⁵ in T. thermophilus HB8 CMK (Fig. 2). Although the amine nitrogen atom of Lys⁸ is 4.2 Å from the nearest carboxyl oxygen atom of Asp¹²⁵ (Fig. 4b), slightly longer than the 4-Å limit usually used in defining a salt bridge (38), the fundamental mechanism of CMK may be the same as that of MVK. Lys⁸ forms very similar interactions to Lys¹³ in MVK, which may fix the amine group in a suitable position for efficient catalysis. In addition, the conserved side chains around the magnesium binding site of MVK (see above) suggest that a magnesium ion could play the same role in CMK. Thus, both the mevalonate and non-mevalonate pathways seem to have a kinase with very similar reaction mechanism.

Substrate Specificity—Since the substrate specificity of CMK has not been reported previously, both T. thermophilus HB8 and E. coli CMK were assayed for kinase activity using a variety of substrates. Both enzymes showed kinase activity for CDP-ME, but not for ADP-ME and UDP-ME (Fig. 5), showing that the cytosine group in CDP-ME plays an essential role for substrate recognition by CMK. MEP cytidylyltransferase (EC 2.7.7.60 [EC] ) converts MEP to CDP-ME in the presence of CTP at the third step of the non-mevalonate pathway (Fig. 1). This enzyme can utilize ATP or UTP instead of CTP, although with less activity (28). Because CMK phosphorylates CDP-ME selectively, only CDP-ME2P is supplied as substrate for the next step. How CDP-ME is distinguished from related ME-derivatives by CMK remains unclear. Among CMKs from different species, the sequence RXDGYHXLXTXF is highly conserved at the loop between S1 and S2 and the N-terminal region of S2 (Figs. 2 and 3a). Other GHMP superfamily proteins do not retain this sequence. The functional role of this region has not been determined, except that Asp¹⁹ in HSK binds to the substrate (32). Thus, this conserved region is the primary candidate as the region contributing of specificity for the cytidine moiety of CDP-ME. Docking CDP-ME onto the enzyme by eye shows that the region may well recognize the cytidine moiety of the substrate (Fig. 3b). Mutational analysis are underway to test this hypothesis.

View larger version (118K): [in this window] [in a new window]   FIG. 5. Assay of CMK activity with CDP-ME, UDP-ME, and ADP-ME as substrates. Reaction mixtures with CDP-ME as substrate are shown in lane 1 (no enzyme), lane 2, (with T. thermophilus HB8 CMK) and lane 3 (with E. coli CMK). Equivalent reactions with UDP-ME as substrate are shown in lanes 4–6, and those with ADP-ME are shown in lanes 7–9, respectively. The autoradiogram of the thinlayer chromatography plate shows a shift in the mobility of the radio-label only in the case of CDP-ME (lanes 2 and 3).

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1UEK [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-45-508-7229; Fax: 81-45-508-7366; E-mail: park{at}tsurumi.yokohama-cu.ac.jp.

¹ The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; CDP-ME, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol 2-phosphate; ADP-ME, 4-(adenosine 5'-diphospho)-2-C-methyl-D-erythritol; UDP-ME, 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol; CMK, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase; HSK, homoserine kinase; MVK, mevalonate kinase; MEP, 2-C-methyl-D-erythritol-4-phosphate; PDB, Protein Data Bank.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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