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J. Biol. Chem., Vol. 277, Issue 11, 9462-9467, March 15, 2002
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,
From the Department of Biochemistry and Biophysics,
Texas A&M University, College Station, Texas 77843-2128 and
¶ Department of Chemistry, Texas A&M University, College Station,
Texas 77843-3255
Received for publication, November 9, 2001, and in revised form, December 18, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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The mevalonate-dependent pathway is
used by many organisms to synthesize isopentenyl pyrophosphate, the
building block for the biosynthesis of many biologically important
compounds, including farnesyl pyrophosphate, dolichol, and many
sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl
transfer step, producing mevalonate 5'-phosphate. The crystal structure
of thermostable MVK from Methanococcus jannaschii has been
determined at 2.4 Å, revealing an overall fold similar to the
homoserine kinase from M. jannaschii. In addition,
the enzyme shows structural similarity with mevalonate 5-diphosphate
decarboxylase and domain IV of elongation factor G. The active site of
MVK is in the cleft between its N- and C-terminal domains. Several
structural motifs conserved among species, including a
phosphate-binding loop, have been found in this cavity.
Asp155, an invariant residue among MVK sequences, is
located close to the putative phosphate-binding site and has been
assumed to play the catalytic role. Analysis of the MVK model in the
context of the other members of the GHMP kinase family offers the
opportunity to understand both the mechanism of these enzymes and the
structural details that may lead to the design of novel drugs.
The ability to make isopentenyl pyrophosphate is essential for all
organisms, because this molecule is the building block for synthesizing
many biologically important compounds, such as ubiquinone, cholesterol,
dolichol, steroid hormones, and some vitamins (1). Two metabolic
pathways, either mevalonate-dependent or -independent, are
available for the production of isopentenyl pyrophosphate. For
mevalonate-dependent isoprenoid synthesis, assembly begins
with the condensation of three acetyl-CoAs to generate a
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-hydroxy--methylglutaryl-CoA molecule, which is reduced to
mevalonate. Mevalonate kinase transfers the 
-phosphoryl group from
ATP to the 5-hydroxyl oxygen in mevalonate to produce phosphomevalonate (Fig. 1). A second phosphoryl group is
then added by phosphomevalonate kinase and the product,
5-pyrophosphomevalonate, is decarboxylated to isopentenyl pyrophosphate
(1, 2). Despite the existence of a mevalonate-independent pathway in
some microorganisms, the process that generates isopentenyl
pyrophosphate via mevalonate is critical in most eukaryotes and some
microorganisms.
View larger version (9K):
[in a new window]
Fig. 1.
The reaction catalyzed by MVK.
Kinases have been classified mainly into three distinct families
according to their sequence similarities and structural folds (3). The
classical P-loop-containing kinases share a common loop between a
-strand and an 
-helix, with the consensus sequence Gly-Xaa2-Gly-Xaa-Gly-Lys (3, 4). A second group, composed primarily of protein kinases (4, 5), has a conserved loop connecting
two antiparallel -strands that are involved in phosphate binding. The third group, which includes actin, Hsp70, and sugar kinases (4, 6), uses two -hairpins to bind the phosphate in ATP.
Mevalonate kinase is a member of the distinct GHMP superfamily, which
includes galactokinase (G), homoserine kinase (H), mevalonate kinase
(M), and phosphomevalonate kinase (P) (4, 7).
4-Diphosphocytidyl-2C-methylerythritol kinase, mevalonate
5'-diphosphate decarboxylase, and archaeal shikimate kinase have also
been assigned as members of this family (4, 8). It is important to note
that shikimate kinases classified in yeast and prokaryotes have the
P-loop kinase fold (8, 9). These proteins share several structural
features. A common motif, Pro-Xaa3-Gly-Leu-Gly-Ser-Ser-Ala-Ala, is highly conserved
and is thought to be involved in the binding of ATP, functioning as a
phosphate-binding loop. The crystal structure of the homoserine kinase,
which shares about 20% sequence identity with the mevalonate kinase,
has recently been solved, both as the apoprotein and as complexed forms (4, 10). Its structure shows a novel left-handed fold similar to domain IV of elongation factor G (4). Similar to classic P-loop kinases, the phosphate binding site is composed of a
conserved loop between a -strand and an -helix, with the consensus sequence Pro-Xaa3-Gly-Leu-Gly-Ser-Ser-Ala-Ala.
Unexpectedly, the
HSK1-substrate complex
structures suggest that the orientation of the bound ATP to HSK is
different from classical P-loop kinases, with the purine base located
at what would be the phosphoryl acceptor substance binding site in a
classic P-loop kinase (3-5, 10). Moreover, ATP seems to adopt an
energetically less favorable syn conformation, which is not
found in classic P-loop kinases (4, 10). The high degree of sequence
similarity in GHMP proteins suggests that other members may share at
least some of the structural features of HSK (4,7).
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Protein Expression and Purification--
The Methanococcus
jannaschii MVK sequence was initially cloned into pET28a vector
with an N-terminal His tag and a T7 promoter, and the construct was
used to transform B834(DE3) Escherichia coli strain (2).
Cells were grown in LB medium containing 100 µg/ml kanamycin at
37 °C until A600 was around 0.8. Cells were harvested, washed with M9 minimal medium (with 100 µg/ml
kanamycin and 50 µg/ml 19 amino acids except Met), and then grown in
this medium for 1 h at 37 °C. Selenomethionine was added to 50 µg/ml and isopropyl -D-thiogalactopyranoside to 1 mM concentration. Cells were grown at 18 °C for 20 h and then harvested and frozen at 
20 °C. Cells were thawed,
re-suspended in 20 mM Tris-HCl, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 20 mM
imidazole (pH 8.0), and then lysed by French press and sonication. The
extract was heated to 70 °C for 15 min to precipitate most E. coli proteins. The enzyme was purified to homogeneity, as judged
by SDS-PAGE, on a nickel affinity column (AP Biotech) following the
instructions of the supplier.
Crystallization and Data Collection--
Crystallization was
accomplished by the hanging drop vapor diffusion method. The MVK (10 to
15 mg/ml) in 5 mM Tris, 10 mM dithiothreitol
(pH 8.0) was mixed with an equal volume of the precipitation solution
(28% dioxane and 10 mM dithiothreitol) and equilibrated
against 100-200 µl of reservoir solution in which components were
the same as the precipitation solution. Single crystals grew overnight
at 16 °C but were generally very small. Well formed crystals were
picked and transferred to newly set protein drops in the same
precipitation solution except with the addition of 10 mM
MgCl2. Larger crystals grew to a size of about 0.2-0.3 mm
in three dimensions. All crystals belong to the space group
P21 with cell dimensions a = 56.138 Å, b = 44.028 Å, c = 64.845 Å, = 90°, = 102.71°,
= 90°, with one molecule in each asymmetric unit.
Data were collected at 121 K using a cryoprotection solution containing the original precipitant with additional 20% 2-methyl-2,4-pentanediol. Data were collected at beamline 14-BM-D at the Advanced Photon Source in Argonne National Laboratory. A four-wavelength (peak, edge, high energy remote, and low energy remote) MAD data set was collected from a single crystal (Table I). 1° oscillation widths were collected through a range of 360° for each wavelength. Data were integrated and reduced using DENZO (11) and SCALEPACK (11).
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Phasing and Refinement-- The structure was solved by the multiple wavelength anomalous dispersion (MAD) method. SOLVE (12-17) was used to determine the heavy atom position and calculate the initial phases. All six selenium sites were found by this method. Phases were improved by using density modification with RESOLVE (18, 19). The map quality was sufficiently good to enable the complete model to be built in the program SPOCK (20). CCP4 programs, including FFTBIG and PROCHECK, have been used to generate the density map and check the final structure quality (21). Refinement was carried out with CNS (crystallographic + NMR systems) (22). The current refinement statistics are listed in Table II.
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Structure Analysis--
DALI (23) was used to search the Protein
Data Bank for proteins having folds similar to the MVK N- and
C-domains. SwissPDB Viewer (24) was used to make structural alignments.
The mevalonate molecule was generated and docked into the MVK cavity
using the AFFINITY module in the INSIGHT program (25, 26). The
ATP was initially modeled into the MVK cavity by superimposing the
HSK-AMPPNP-homoserine structure with the apo-MVK structure and changing
AMPPNP to ATP (9). The conformation of ATP is the same as that of
AMPPNP; however, the ATP molecule was rotated slightly and translated to achieve a low energy between the ATP and the protein, calculated using INSIGHT. All other analysis of the structure and all figures shown were produced using SPOCK (20).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Overall structure of MVK--
Three very conserved motifs,
designated motif I, II, and III, have been identified in GHMP proteins
(4). The multiple sequence alignment of MVK from different
species and the M. jannaschii HSK is shown in Fig.
2. Motif I is close to the N terminus of the protein, including residues corresponding to Lys8 and
Glu14 of the M. jannaschii MVK. Motif II
corresponds to the region between Pro104 and
Ser114 of the M. jannaschii MVK, with an
internal Gly-Leu-Gly-Ser-Ser-Ala sequence that is invariant, and forms
the phosphate-binding loop. Motif III is characterized by
Lys272-Cys281 of the Methanococcus
jannaschii MVK and has an invariant sequence Thr-Gly-Ala-Gly-Gly-Gly-Gly among various mevalonate kinases. In HSK,
this region corresponds to the sequence Thr-Ile-Ser-Gly-Ser-Gly-Pro, a
loop in close contact with the phosphate-binding loop, which is
presumed to stabilize the conformation of the latter (4).
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The structure of MVK contains all 312 residues of the wild-type MVK as well as five residues from the N-terminal histidine tag fusion. It is a monomer in crystal, although the dimer was reported in the aqueous solution (2). The R-factor has been refined to 0.20 with an accompanying Rfree value of 0.28 (Table II). Nearly 88% of the non-glycine residues are in the most favorable conformation.
The C
of MVK is shown in Fig.
3a. Similar to HSK, the MVK
monomer contains two structural domains. At the domain interface there
is a deep cavity presumed to be the active site (Fig. 3, a
and b). Two molecules of dioxane, used as the
crystallization precipitant, have been found to occupy this cavity
(Fig. 5b). The structure of MVK is readily superimposable
with that of HSK (Fig. 3c), indicating the structural
conservation among the GHMP kinases. The root mean square deviation is
1.6 Å between the 155 superimposed C atoms.
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The Structure of the N-terminal Domain and the Fold
Comparison--
The N-terminal domain of MVK comprises a large mixed
five-stranded -sheet (formed by strand a, b, c, d, and e) and a
smaller anti-parallel four-stranded -sheet (formed by strand a, b,
f, and g), which are packed with four long -helices (Fig.
4a).
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The coordinates for the N-terminal domain (defined as residues 1-176) were used to search for similar structures by DALI (23). The best match was with 1fwk, the structure of HSK (4), with a Z-score 16.6. The second highest hit was 1fi4, the mevalonate 5-diphosphate decarboxylase (MDD) from Saccharomyces cerevisiae (27), with a Z-score of 9.7. Other top hits include 1dar, the elongation factor G (Z-score 5.4); 1b63, the Mutl protein (Z-score 5.0); and 1e3h, the guanosine pentaphosphate synthetase (Z-score 4.2).
Although the N-terminal domains of MVK, HSK, and MDD (shown in Fig. 4,
a-c) are similar, there are substantial differences around
the region corresponding to -strand c in MVK. Compared with HSK,
there is an insertion between -strand c and -helix D in MVK,
which includes strand d and most of the following loop. MDD lacks the
equivalent -strands c and d of MVK, but instead it has a very long
loop that links -strand f (equivalent to strand b in MVK) with
-helix A (corresponding to helix D in MVK).
The Structure of the C-terminal Domain and the Fold
Comparison--
The structure of the C-terminal domain has a core unit
that adopts a fold, formed with a four-strand
-sheet (strand h, i, j, and k) packed with two helices (L and
M). There are three additional helices (I, J, and K) that are
considered to be an insert between strand h and helix L (4).
DALI searches were performed using the coordinates of the C-terminal domain (defined as residues 177-312). The top match corresponds to 1fwk, the code for homoserine kinase (HSK), with a Z-score of 9.9, followed by 1fi4, the mevalonate 5-diphosphate decarboxylase, with Z-score 9.4. All other hits possessed Z-scores less than 5.0, including 1dar (elongation factor G, Z score 4.2), 1ha1 (a heterogeneous nuclear ribonucleoprotein, Z-score 4.0), and many others.
The C-terminal domains of MVK, HSK, and MDD are shown in Fig. 4,
d-f. Each possesses a similar core unit with several
insertions composed mainly of -helices. Compared with those in HSK
and MDD, the C-terminal tail in MVK is truncated and lacks the
C-terminal -strand (strands l and n in HSK and MDD, respectively)
that folds toward the N-terminal domain and joins the large mixed
-sheet.
Homology Model of the Active Site of MVK-- Mutations such as E19D, S145A, S146A, E193Q, D204A, and A334T in human MVK and K13M in rat MVK have been shown to decrease the activity of the enzymes (28-31). In M. jannaschii MVK, Glu14, Ser111, Ser112, Glu144, Asp155, Ala276, and Lys8 are identical to the equivalent residues of the human and rat protein, respectively (Fig. 2). Among the mutations studied, D204A elicits the most pronounced effects on catalysis by decreasing Vm ~10,000-fold (28). These results helped to establish the probable mechanism of the enzyme and facilitated our analysis of the active cavity.
All of these conserved residues are located in a region that defines
the active site cavity (Fig.
5a). For example, the residues corresponding to motif III form a Gly-rich loop very close to the
phosphate-binding loop corresponding to motif II. Cys281 in
the Gly-rich loop is ~2.0 Å from Cys107 in the
phosphate-binding loop. The relative orientation of these two residues
as well as a continuous electron density connecting their side chains
suggest a disulfide bond, despite the existence of dithiothreitol in
the crystallization condition. Formation of the disulfide linkage may
stabilize the conformation of the putative phosphate-binding loop. In
addition, Lys272 in the Gly-rich loop is 3.1 Å away from
Glu14, a motif I residue, which indicates electrostatic
interactions.
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The positions of the phosphate-binding loops of MVK and HSK (Fig. 5,
b and c) are nearly identical when the proteins
are superimposed. A dioxane molecule is bound about 3.0 Å from
the main chain amides of Ser111 and Ser112
(which are both invariant residues on the phosphate-binding loop) and
probably forms hydrogen bonds with these two amino acids. In various
HSK-substrate complex structures, this region is occupied by phosphate
groups and hydrogen bonds with the main chain amides on the
phosphate-binding loop are present. For example, one HSK-ADP structure
shows that the -phosphate group occupies this position and forms
hydrogen bonds with the main chain amides of Ser97 and
Ser98 (equivalents of Ser111 and
Ser112). Close to the phosphate-binding loop is the
invariant residue Glu144, which aligns with
Glu130 in HSK, a residue that coordinates the
Mg2+ ion (10). These results reveal roughly the region in
MVK where the phosphate tail of ATP may bind. In HSK, the purine ring
is bound in a pocket close to the phosphate-binding site. A similar pocket is apparent in MVK that likely serves the same function. The
backbone of Lys74-Cys76 of MVK can be
superimposed to the backbone of
Asn62-Ala64 of HSK. In HSK, these residues
form van der Waals contacts with the purine ring (within 5 Å), and the
main chain amide of Val63 forms a hydrogen bond with
N7 in the purine. Although the exact amino acids are
somewhat different, the similarity of the molecular surfaces and the
constellation of protein atoms in this region suggest that it is likely
that in MVK these corresponding residues may form similar interactions with the purine ring.
Based on the alignment of the structures of apo-MVK and
HSK-AMPPNP-homoserine, an ATP molecule and a mevalonate molecule were docked into the cavity (Fig. 5d). The phosphate tail of the
docked ATP molecule closely interacts with the phosphate-binding loop (within 5 Å) and forms hydrogen bonds with several main chain amides,
including that of Ser112. The purine ring fits well into
the putative purine-binding pocket, consistent with the analysis
described above. The carboxyl group of the docked mevalonate molecule
is near a large positively charged patch in the C-domain part of the
cavity and forms hydrogen bonds with the main chain amide of
Ala276 and the side chain amide of
Arg196. The 3'-methyl group of the mevalonate fits into a
hydrophobic patch at the N-domain side of the cavity. In the model, the
3'-OH group is within hydrogen bond distance of the side chain of
Lys8, a conserved residue in motif I. The 5'-OH group in
the docked mevalonate is less than 3.0 Å from the -phosphorus of
the docked ATP molecule. The 5'-OH group of the modeled mevalonate is
only about 4.0 Å from Asp155. The model indicates a
possible reaction mechanism that is consistent with previous
enzymological studies on MVK.
Possible Reaction Mechanism--
The reaction is likely to be
initiated by the nucleophilic attack to the -phosphorus of ATP by
the 5'-OH of mevalonate (Fig. 6). Based
on the analysis of the active cavity, the best candidate for the
catalytic residue is Asp155 (located about 4.0 Å from the
5'-OH of the docked mevalonate), which is consistent with the mutation
data available for human MVK (28). It is likely that Asp155
functions as a general base with its side carboxyl group in position to
abstract a proton from the 5'-OH of mevalonate, thereby facilitating the nucleophilic attack. Notably, this residue is structurally aligned
with Asn141 in HSK, which has been shown to interact
directly with the 
-OH of the homoserine (10). Lys8,
Ala276, and Arg196 appear to be key to the
binding of mevalonate (Fig. 6). The phosphate-binding loop might be the
main force to bind and stabilize the phosphate tail of ATP. The N
terminus of -helix E is pointing toward the phosphate groups and may
offer some stabilizing effect during phosphoryl transfer, as is the
case in many other kinases (3,4). The Mg 2+ that
might be coordinated by Glu144 is likely to activate the
-phosphate. This mechanism is consistent with the model proposed by
the Miziorko laboratory (29).
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In conclusion, our work has defined the structure of M. jannaschii mevalonate kinase and revealed the structural
conservation among GHMP proteins. As more GHMP protein structures are
solved, the detailed comparison and analysis of these proteins with
substrates and inhibitors will further our understanding of the
enzymatic mechanisms and offer us opportunities to design potential
drugs against various diseases.
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ACKNOWLEDGEMENTS |
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We thank Advanced Photon Source personnel for assistance at beamline 14-BM-D.
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FOOTNOTES |
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* Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the Office of Energy Research, United States Department of Energy, under Contract W-31-109-ENG-38. This work was supported by the Robert A. Welch Foundation and by a grant from the National Institutes of Health.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 1KKH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Current address: Dept. of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
To whom correspondence should be addressed. Tel.:
979-862-7636; Fax: 979-862-7638; E-mail: sacchett@tamu.edu.
Published, JBC Papers in Press, December 19, 2001, DOI 10.1074/jbc.M110787200
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ABBREVIATIONS |
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The abbreviations used are:
HSK, homoserine
kinase;
MVK, mevalonate kinase;
MAD, multiple wavelength anomalous
dispersion;
AMPPNP, adenosine 5'-(,-imino)triphosphate or
adenyl-5'-yl iminodiphosphate;
GHMP, galactokinase, homoserine kinase,
mevalonate kinase, and phosphomevalonate kinase;
MDD, mevalonate
5-diphosphate decarboxylase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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A. K. Nair and K. M. J. Menon Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary J. Biol. Chem., April 9, 2004; 279(15): 14937 - 14944. [Abstract] [Full Text] [PDF]
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M. Hedl and V. W. Rodwell Enterococcus faecalis mevalonate kinase Protein Sci., March 1, 2004; 13(3): 687 - 693. [Abstract] [Full Text] [PDF]
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 N. E. Voynova, S. E. Rios, and H. M. Miziorko Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway J. Bacteriol., January 1, 2004; 186(1): 61 - 67. [Abstract] [Full Text] [PDF]
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H. M. Holden, I. Rayment, and J. B. Thoden Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism J. Biol. Chem., November 7, 2003; 278(45): 43885 - 43888. [Full Text] [PDF]
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J. B. Thoden and H. M. Holden Molecular Structure of Galactokinase J. Biol. Chem., August 29, 2003; 278(35): 33305 - 33311. [Abstract] [Full Text] [PDF]
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T. Wada, T. Kuzuyama, S. Satoh, S. Kuramitsu, S. Yokoyama, S. Unzai, J. R.H. Tame, and S.-Y. Park Crystal Structure of 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, an Enzyme in the Non-mevalonate Pathway of Isoprenoid Synthesis J. Biol. Chem., August 8, 2003; 278(32): 30022 - 30027. [Abstract] [Full Text] [PDF]
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 L. Miallau, M. S. Alphey, L. E. Kemp, G. A. Leonard, S. M. McSweeney, S. Hecht, A. Bacher, W. Eisenreich, F. Rohdich, and W. N. Hunter Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase PNAS, August 5, 2003; 100(16): 9173 - 9178. [Abstract] [Full Text] [PDF]
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 J. G. Luz, C. A. Hassig, C. Pickle, A. Godzik, B. J. Meyer, and I. A. Wilson XOL-1, primary determinant of sexual fate in C. elegans, is a GHMP kinase family member and a structural prototype for a class of developmental regulators Genes & Dev., April 15, 2003; 17(8): 977 - 990. [Abstract] [Full Text] [PDF]
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D. Pilloff, K. Dabovic, M. J. Romanowski, J. B. Bonanno, M. Doherty, S. K. Burley, and T. S. Leyh The Kinetic Mechanism of Phosphomevalonate Kinase J. Biol. Chem., February 7, 2003; 278(7): 4510 - 4515. [Abstract] [Full Text] [PDF]
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Z. Fu, M. Wang, D. Potter, H. M. Miziorko, and J.-J. P. Kim The Structure of a Binary Complex between a Mammalian Mevalonate Kinase and ATP. INSIGHTS INTO THE REACTION MECHANISM AND HUMAN INHERITED DISEASE J. Biol. Chem., May 10, 2002; 277(20): 18134 - 18142. [Abstract] [Full Text] [PDF]
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hkl
I(hkl)
|/
around the side chain of
Cys107 and Cys281, showing a possible disulfide
bond. The amino acid residues are colored according to the atoms:
white, carbon; red, oxygen; yellow,
sulfur; blue, nitrogen. b-d show the
water-accessible surface and its electrostatic potential for MVK: HSK
(b) and MVK (c) with docked ATP and mevalonate
(d). The positively charged region is colored
blue and the negative region red. The
amino acid residues, ADP, ATP, mevalonate (MEV), and dioxane
molecules are colored as described in a. The
phosphate-binding loop is shown in yellow. The N terminus of
helix D (in MVK) or helix A (in HSK) is shown in white. In
c, residues Thr183 and Ser134 have
been changed to Gly to remove a bridge across the active pocket so that
the pocket can be revealed fully in the figure.
