Journal of Biochemistry and Molecular Biology, Vol. 40, No. 6, November 2007, pp. 911-920
Biosynthesis of Isoprenoids: Characterization of a Functionally Active
Recombinant 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase (IspD)
from Mycobacterium tuberculosis H37Rv
Wenjun Shi1,2, Jianfang Feng3, Min Zhang1, Xuhui Lai1, Shengfeng Xu1, Xuelian Zhang1,* and Honghai Wang1,*
1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University,
220 Handan Road, Shanghai 200433, P. R. China
2Medical School, Tongji University, 1239 Siping Road, Shanghai, 200092, P. R. China
3Pharmaceutical Division, Shanghai Institute of Pharmaceutical Industry, 1111 Zhong Shan No.1 Road (N), Shanghai, 200437, P. R. China
Received 22 May 2007, Accepted 4 July 2007
Tuberculosis, caused by Mycobacterium tuberculosis,
continues to be one of the leading infectious diseases to
humans. It is urgent to discover novel drug targets for the
development of antitubercular agents. The 2-C-methyl-D-
erythritol-4-phosphate (MEP) pathway for isoprenoid
biosynthesis has been considered as an attractive target for
the discovery of novel antibiotics for its essentiality in
bacteria and absence in mammals. MEP cytidyltransferase
(IspD), the third-step enzyme of the pathway, catalyzes
MEP and CTP to form 4-diphosphocytidyl-2-C-methyl-
erythritol (CDP-ME) and PPi. In the work, ispD gene from
M. tuberculosis H37Rv (MtIspD) was cloned and expressed.
With N-terminal fusion of a histidine-tagged sequence,
MtIspD could be purified to homogeneity by one-step
nickel affinity chromatography. MtIspD exists as a
homodimer with an apparent molecular mass of 52 kDa.
Enzyme property analysis revealed that MtIspD has high
specificity for pyrimidine bases and narrow divalent cation
requirements, with maximal activity found in the presence
of CTP and Mg2+. The turnover number of MtIspD is 3.4 s−1.
The Km for MEP and CTP are 43 and 92 µM, respectively.
Furthermore, MtIspD shows thermal instable above 50oC.
Circular dichroism spectra revealed that the alteration of
tertiary conformation is closely related with sharp loss of
enzyme activity at higher temperature. This study is
expected to help better understand the features of IspD
and provide useful information for the development of
novel antibiotics to treat M. tuberculosis.
Keywords: Circular dichroism, Enzyme, Mycobacterium
tuberculosis, MEP pathway, MEP cytidyltransferase
Tuberculosis (TB), infected with Mycobacterium tuberculosis,
remains the leading infectious disease to humans. It accounts
for approximately 8 million new cases worldwide and an
estimated 2 million deaths annually. The emergence of multi-
drug resistant strains and their synergy with HIV infection
have fuelled spread of the disease. Accordingly, there is an
urgent need to identify new drug targets and develop
inhibitors against the pathogen.
Isopentenyl diphosphate (IPP) or its isomer dimethylallyl
diphosphate (DMAPP) is the common precursor in the synthesis
of various isoprenoid compounds. IPP was traditionally known to
be synthesized from the mevalonate pathway, which begins with
the condensation of three molecules of acetyl-CoA. However, the
existence of an alternative pathway, known as the 2-C-methyl-D-
erythritol 4-phosphate (MEP) pathway, was established relatively
recently. The enzymes and intermediates of the MEP pathway are
completely distinct from those of the classical mevalonate
pathway (Fig. 1). The pathway utilizes pyruvate (1) and D-
glyceraldehyde 3-phosphate (2) as starting materials to yield 1-
deoxy-D-xylulose 5-phosphate (3, DXP) (Sprenger et al., 1997;
Lois et al., 1998). This intermediate is reductively rearranged to
Abbreviations: MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-
ME, 4-diphosphocytidyl-2-C-methylerythritol; MtIspD, IspD from
Mycobacterium tuberculosis; EcIspD, IspD from Escherichia coli;
CjIspDF, IspDF from Campylobacter jejuni; ScIspD, IspD from
Streptomyces coelicolor; AtIspD, IspD from Arabidopsis thaliana;
IspDF from Mesorhizobium loti; AtIspDF, IspDF from Agrobacte-
rium tumefaciens; IPTG, isopropyl-β-D-thiogalactopyranoside; CD,
*To whom correspondence should be addressed.
Tel: 86 21 65643777; Fax: 86 21 65648376
E-mail: firstname.lastname@example.org (H. Wang)
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912Wenjun Shi et al.
2-C-methyl-D-erythritol 4-phosphate (4, MEP) by DXP
reductoisomerase (DXR or IspC) (Kuzuyama et al., 1998b).
MEP is further converted to 4-diphosphocytidyl-2-C-methyl-
erythritol (5, CDP-ME) by MEP cytidyltransferase (IspD)
(Rohdich et al., 1999; Kuzuyama et al., 2000a). In the following
reaction steps, CDP-ME is phosphorylated by CDP-ME kinase
(IspE) to produce 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-
phosphate (6, CDP-MEP) (Kuzuyama et al., 2000b; Luttgen et
al., 2000), which is cyclized to 2-C-methyl-D-erythritol-2, 4-
cyclodiphosphate (7, MECP) by MECP synthase (IspF) (Herz et
al., 2000; Takagi et al., 2000). The ring of the cyclodiphosphate 7
is reductively opened to yield 1-hydroxy-2-methyl-2(E)-butenyl-
4-diphosphate (8) (Hecht et al., 2001; Seemann et al., 2002;
Altincicek et al., 2001b). Finally, LytB (IspH) tranforms 8 to a
mixture of IPP (9) and DMAPP (10) (Cunningham et al., 2000;
Altincicek et al., 2001a; Rohdich et al., 2002).
It has been fully established that the MEP pathway is the only
source of isoprenoids in a number of eubacteria, green algae,
chloroplasts of higher plants and apicoplasts of apicomplexan
parasites (Rohmer et al., 1993; Jomaa et al., 1999). The
essential nature of the MEP pathway in these organisms and
absence in mammals make enzymes of the pathway attractive
targets for the development of herbicides, antibiotics and
antimalarial drugs (Freiberg et al., 2001; Cornish et al., 2006;
Estévez et al., 2000). So far, the antibiotic fosmidomycin and
its derivative of FR 900098 have been demonstrated to inhibit
the second-step enzyme of the MEP pathway (IspC) and
perform well in clinical trials against Plasmodium falciparum
(Kuzuyama et al., 1998a; Lell et al., 2003).
In mycobacteria, the MEP pathway leads to the precursors of
menaquinone and polyprenyl phosphates (Pol-P). Menaquinone
is the electron transporter in mycobacterial respiratory chain
(Jones et al., 1975). Pol-P are building blocks for the special
cell wall components of mycobacteria, such as peptidoglycan-
arabinogalactan-mycolic acid complex as well as lipomannan
and lipoarabinomannan (Wolucka et al., 1994). Thus, the
reactions involved in the MEP pathway should represent
excellent potential target sites for chemotherapy against M.
tuberculosis. The properties of the enzymes involved in the
pathway would provide important information for the design
of novel antitubercular agents.
MEP cytidyltransferase (IspD, EC 184.108.40.206) is the third-step
enzyme of the MEP pathway, and catalyzes MEP and CTP to
form CDP-ME and PPi. The encoding gene ispD neighbours
ispF in the genomes, which is fairly different from dispersed
distribution of other genes in the MEP pathway. Gene IspD
and IspF are transcriptionally coupled or, in a few bacteria
species, encode together for a bifunctional enzyme (Gabrielsen
et al., 2004b). This bifunctional IspDF enzyme is quite special
because it catalyzes nonconsecutive steps in the pathway. It
implied that IspD had physical association with both IspE and
IspF, which may have a role of regulation and organization of
the MEP pathway. Thus, biophysical and biochemical properties
of IspD would be helpful for deciphering the mechanisms and
designing drugs. So far, IspD has been cloned and partially
characterized from several sources (Rohdich et al., 1999;
Cane et al., 2001; Rohdich et al., 2000), only gram-negative
Campylobacter jejuni is pathogen (Gabrielsen et al., 2004b).
Specially, C. jejuni produces a bifunctional IspDF. No
information about monofuctional IspD from other pathogenic
bacterium has been characterized. According to the genome
annotation, pathogenic M. tuberculosis H37Rv IspD and IspF
genes encode a monofuctional protein, respectively. In this
report, we cloned and expressed M. tuberculosis H37Rv IspD
Fig. 1. The MEP pathway of isoprenoid biosynthesis.
Properties of 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase from M. tuberculosis913
gene (Rv3582c) in E. coli system, and biochemical and
enzymatic characterizations of the recombinant protein were
determined. This study is expected to help better understand
the features of monofuctional IspD from pathogen, and
provide useful information for the development of novel
antibiotics to treat M. tuberculosis.
Materials and Methods
Materials. All chemicals were purchased from Sigma Chemical Co
unless stated otherwise. MEP was from Echelon Biosciences.
Plasmid pET28a expression vector, E. coli DH5α and BL21(DE3)
strains were obtained from Novagen. The genome of M. tuberculosis
H37Rv was from our lab.
Cloning of M. tuberculosis IspD gene. The coding region of the
IspD gene from M. tuberculosis H37Rv genome was amplified by
PCR with the following primers: Forward, 5'-TGGAACTCATATG
GTCAGGGAAGCGGGC-3'; Reverse, 5'-CCTCTCGAGTTCACC
CGCGCACTATAGCT-3'. The NdeI and XhoI sites (underlined)
were introduced, respectively. The PCR reaction was performed as
follows: 94oC (2 min) for 1 cycle, followed by 94oC (15 s) and 68oC
(75 s) for 25 cycles, and 68oC (2 min) for 1 cycle. The ~700 bp
amplification was purified and digested with NdeI and XhoI
endonucleases (TaKaRa), then subcloned into the plasmid pET28a.
The ligation mixture was transformed into E. coli DH5α, and
positive colonies were screened on LB agar plates in the presence
of 50 µg/ml kanamycin. Constructs possessing the correct insert
were chosen on the basis of restriction digests with NdeI/XhoI, and
confirmed by automated DNA sequencing using ABI 377 analyzer
(Applied Biosystems). The desired plasmid was designated as
Sequence analysis. Blast analysis shows similarities between MtIspD
orthologs. Multiple alignments were performed among IspD sequences
from major pathogenic bacteria using ClustalW at http://www.ebi.
ac.uk/clustalw/. The output of alignment was edited with the
GeneDoc program. The phylogenetic tree was constructed using
MEGA 3.1 with the neighbor-joining (NJ) method. Sequences were
selected mainly from those that the recombinant IspD proteins had
been reported. In the analysis, the gaps were deleted, and a 1000
bootstrap procedure was used to test the robustness of the node on
the tree. The secondary structure prediction was done using the
program JPRED (http://www.combio.dundee.ac.uk/~www-jpred/)
and GOR 4 (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=
Expression and purification of MtIspD. The plasmid pET28a-
MtIspD was transformed into competent E. coli BL21(DE3).
Bacteria cells were grown in LB broth supplemented with 50 µg/ml
kanamycin. The culture was incubated at 37oC with vigorous
shaking. At an optical density (600nm) of 0.5~0.6, IPTG was added
to a final concentration of 1mM, and the culture was further incubated
at 16oC for 12 h. The cells were harvested by centrifugation and
stored at −80oC. Cell pellets were thawed and re-suspended in lysis
buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM imidazole)
containing 1 mg/ml lysozyme. The mixture was incubated at 4oC
for 30 min and subsequently sonicated on ice. The crude lysate was
centrifuged at 16,000 rpm at 4oC for 60 min to remove cellular
debris. The supernatant was loaded on a Ni-NTA resin column
(Novagen) pre-equilibrated with lysis buffer. The column was
washed with 20 bed volumes of wash buffer A (50 mM Tris-HCl,
pH 8.0, 300 mM NaCl, 20 mM imidazole), 20 bed volumes of
wash buffer B (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 50 mM
imidazole) and 2 bed volumes of wash buffer C (50 mM Tris-HCl,
pH 8.0, 300 mM NaCl, 100 mM imidazole). The recombinant
protein was eluted with elution buffer (50 mM Tris-HCl, pH 8.0,
300mM NaCl, 200mM imidazole). The eluted protein was dialyzed
overnight against 5 mM Tris-HCl, pH 8.0 and concentrated by
ultrafiltration using a 10-kDa molecular mass cutoff filter (Millipore).
The protein concentration was determined by Bradford method
using bovine serum albumin (BSA) as standard. The purified
protein was divided into aliquots before storage at −80oC. All the
purification procedures were conducted at 4oC.
Mass spectral investigation of MtIspD. The LC/MS system
comprises an HP 1100 LC system (HP) with an API 165 mass
spectrometer (PE). 100 µl purified MtIspD sample was separated
with a reverse phase column C18 (4.6 × 250 mm) (Agilent) using
0.1% trifluoroacetic acid (TFA) followed by an increasing linear
gradient of 80% isopropanol in 0.085% TFA at a flow rate of 1.0
ml/min. Fractions were collected across the peak and analyzed by
mass spectrometry. The scan mass range was from m/z 300 to 3000.
Native Gel Electrophoresis of MtIspD. The native molecular
mass of MtIspD was evaluated using nondenaturing electrophoresis.
Protein standards were α-lactalbumin (14.2kDa), carbonic anhydrase
(29kDa), chicken egg albumin (45kDa), and bovine serum albumin
(monomer, 66 kDa, and dimer, 132 kDa). Protein standards and
purified MtIspD were electrophoresed on a set of nondenaturing
gels with polyacrylamide concentrations of 6, 7, 8, 9, or 10%,
respectively. Electrophoresis was carried out at 80 V for 40 min and
at 140 V for 2 h. The tracking dye was bromophenol blue. Proteins
were visualized after staining with Coomassie blue R-250. The
relative mobility (Rf) of the protein was determined by the distance
of the protein migration relative to the tracking dye migration. A
value of 100 [log (Rf× 100)] for each protein was plotted against
the percent concentration of acrylamide and the slope of the line
was determined. The negative slope obtained for each protein was
then plotted against the native molecular masses of protein
standards to produce a linear log/log plot from which the molecular
mass of MtIspD was extrapolated.
Enzymatic characterization of MtIspD. MtIspD catalyzes MEP
and CTP to form CDP-ME and PPi. Enzyme activity was assayed
by using an inorganic pyrophosphatase-coupled assay as described
by C. Bernal et al. (Bernal et al., 2005). In the method, the product
PPi is further hydrolyzed by inorganic pyrophosphatase to produce
inorganic phosphate, which forms a complex with ammonium
molybdate-malachite green spectrometrically detectable at 630 nm.
All assays were conducted at 30oC in a 96-well plate system. The
standard assay system consisted of 50 mM Tris-HCl, pH 8.0, 2 mM
MgCl2, 2 mM DTT, 1 mM MEP, 1 mM CTP, 0.1 U/ml inorganic
pyrophosphatase and 5 µl enzymes in a final volume of 40 µl. All
of the components except for MtIspD were premixed and pre-
914Wenjun Shi et al.
incubated at 30oC for 10 min to reach a stable background. The
MtIspD was added in the mixture to trigger the reaction. The
reaction was carried out at 30oC for 15 min and terminated by
addition of 40µl ammonium molybdate/malachite green dye reagent
and 120 µl distilled water. This final mixture was equilibrated at
30oC for 10 min and detected at 630 nm using a microtiter plate
reader (Thermo, Waltham, USA). Inorganic phosphate production
was quantified by comparing absorbance to calibration curve of
The kinetic parameters were determined according to the published
method. Effects of pH, divalent cations, nucleotide triphosphates
and temperature on enzyme activity were also determined. All
results shown were the average of three different experiments.
Circular dichroism spectrum investigation of MtIspD. All the
circular dichroism (CD) assays were carried out using a JASCO J-
715 spectropolarimeter (Jasco) with a temperature controller (Naslab).
The path lengths were 0.1 cm for far-UV (190-240 nm) and 1 cm
for near-UV (250-320 nm). The MtIspD concentration was 0.2 mg/
ml for far-UV and 2.0mg/ml for near-UV. The effect of temperatures
on MtIspD was investigated from 25oC to 80oC. At each given
temperature, 10 min being allowed for equilibration before the
spectrum was recorded. Five scans were averaged to obtain each
final spectrum. Buffer background was subtracted from the original
spectra. Data were expressed in terms of molar ellipticity [θ] in deg
cm2 dmol−1 s. Secondary structure parameters were determined by
the computer program PROSEC derived by Yang and coworkers
(Yang et al., 1986)
Results and Discussion
Sequence analysis of MtIspD. MtIspD was subjected to
alignment with the predicted sequences of IspD from several
pathogenic bacteria (Fig. 2A). Two conserved motif AAGX
GXRX5PK and [V/I]L[V/I]HDXAR were found in MtIspD.
The glycine-rich motif, AAGXGXRX5PK, coupled with Gly
82, Asp 83 and Ser 88 sequesters CTP and CDP-ME in the
active site (Richard et al., 2001). However, the leeway was
found in IspD sequences from M. tuberculosis and M. bovis,
in which strictly conserved Ser 88 was conservatively
replaced by threonine. (V/I)L(V/I)HDXAR is a characteristic
signature motif of IspD. The key residues of Arg 20, Lys 27,
Arg 157 and Lys 215 conserved in MtIspD are major
contributors to the enzyme mechanism of which the basic side
chains are involved in binding and processing substrates
(Richard et al., 2001; Kemp et al., 2003).
BLAST analysis suggested that sequence identities among
pathogenic bacteria vary from 32% to 99%. The same genus
of mycobacteria shares high sequence identities, eg Mycobacterium
bovis (GI: 31620356, 99% identity), Mycobacterium leprae
TN (GI: 15827085, 67% identity). MtIspD has 34% identities
with EcIspD, similar to 32% with plant AtIspD. Based on
ClustalW and BLAST analysis, phylogenetic tree were
constructed with NJ method (Fig. 2B). The phylogenectic tree
clearly indicated that bacteria IspD can be divided into two
groups. The first group includes actinomycetales species, while
the second group consists of EcIspD, MlIspDF and AtIspDF.
The phylogenetic tree based on the IspD generally revealed
their evolutionary distances of several species, and MtIspD
has much closer phylogenetic relationship with ScIspD than
with EcIspD and other bifuntional IspDF proteins.
Expression and purification of MtIspD. Based on the available
genome sequence of M. tuberculosis H37Rv, cloning of IspD
gene from M. tuberculosis H37Rv was performed. The IspD
gene from M. tuberculosis H37Rv (Rv3582c) begins with a
GTG codon at the initiation site for the translation of IspD
protein. Since ATG, instead of GTG, is an initiation codon for
efficiently translational initiation in the host E. coli BL21(DE3),
we changed the GTG codon of MtispD to ATG by the forward
primer. The amplified gene was subcloned into the NdeI/XhoI
sites of the plasmid pET28a. DNA sequencing confirmed that
the inserted fragment was the putative gene sequence of IspD
The recombinant plasmid pET28a-MtIspD was used for
heterologous expression of MtIspD in E. coli BL21(DE3) strain.
To obtain high level of soluble MtIspD protein, we reduced the
induction temperature to avoid formation of inclusion body.
Optimal expression was obtained with 1mM IPTG at 16oC for
12 h. With N-terminal fusion of a six-histidine tag, the
recombinant MtIspD could be purified to homogeneity by one-
step purification of nickel affinity chromatography. The overall
yield of the purified protein was estimated to be about 2.8mg
from 1l culture medium (Table 1). The turnover number of
MtIspD is 3.4s−1, in close to catalytic rate from the same
actinomycetales species of S. coelicolor and slightly lower than
other bacterial sources and plant A. thaliana (Table 2) (Cane et
al., 2001; Rohdich et al., 1999; Gabrielsen et al., 2004b).
Determination oligomerization of MtIspD. On SDS-PAGE,
the purified MtIspD indicated as a single band with an
apparent molecular mass of ~26 kDa (Fig. 3A). The accurate
molecular mass was further determined by mass spectrometry
(Fig. 3B). The measured mass is 26112.0 Da, in consistent to
the theoretical mass prediction of the protein (the calculated
mass, 26105.66 Da). It thereby demonstrates veracity of the
expressed recombinant MtIspD.
Previous x-ray structure studies presented that IspD organizes
as a homodimer (Richard et al., 2001; Kemp et al., 2003;
Gabrielsen et al., 2006). The core domain of each subunit is
globular in shape and the subdomain resembles a curved arm
to mediate dimer formation. This interlocking arms form part
of the MEP binding site and organize portions of the catalytic
surface responsible for cytidyltransferase activity (Richard et
al., 2001). Thus, we carried out nondenaturing polyacrylamide
gel electrophoresis to investigate the quaternary structure of
MtIspD. Fig. 4 showed two main upper bands, which are
charge isomers and correspondent to a molecular weight
around 52 kDa. Still, there is a very weak band at the lower
site of the gel, which exhibits a molecular weight of 26 kDa.
Properties of 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase from M. tuberculosis915
Fig. 2. Sequence analysis of MtIspD. (A) Sequence alignment of MtIspD with seven bacterium homologies. Sequences were aligned using
ClustalW at http://www.ebi.ac.uk/clustalw/. Identical residues across all sequences are highlighted in black and conservative residues in gray.
The conserved motifs are denoted under the sequences and secondary structures are labeled above. The species are denoted by the
abbreviations as following: Mt, Mycobacterium tuberculosis H37Rv (GI:15610718); Mb, Mycobacterium bovis AF2122/97 (GI:31794759);
Ml, Mycobacterium leprae TN (GI:15827085 ); Cd, Corynebacterium diphtheriae NCTC 13129 (GI:38234537); Ec, Escherichia coli K12
(GI:16130654); St, Salmonella typhi CT18 (GI:16761702); Vc, Vibrio cholerae O1 N16961 (GI:15640032); Hi, Haemophilus influenzae
(GI:68249251). (B) A phylogenetic tree was constructed by MEGA 3.1 among IspD proteins which characterizations have been reported.
The numbers indicate the bootstrap confidence values obtained for each node after 1000 replications.
916Wenjun Shi et al.
Therefore, the recombinant MtIspD is a functionally active
homodimer protein. However, Gabrielsen et al. reported that
EcIspD displays as monomer, dimer and unexpected tetramer
by size exclusion chromatography (Gabrielsen et al, 2004a). It
revealed that this tetramer is formed by dimer-dimer disulfide
linkage of cysteine residue 25. We identified that Cys25 is not
conservative residue among IspD family proteins, and the
corresponding residue in MtIspD is valine 25. Accordingly,
tetramer is not a necessary structure for IspD proteins.
Enzymatic properties of MtIspD. Enzymatic activity was
evaluated by using an inorganic pyrophosphatase-coupled
assay (Bernal et al., 2005). In order to investigate the apparent
Km of recombinant MtIspD, enzyme activity was determined
by varying concentrations of one substrate while keeping
concentrations of the other substrate and the recombinant
enzyme constant (Fig. 5). The apparent Km for MEP and CTP
were 43 and 92 µM, respectively. The recombinant MtIspD
displayed similar affinity for the substrates compared to
EcIspD and CjIspD, as shown by the similar range of their
Km values (Table 2). Interestingly, MtIspD substrate affinities
deviate form the recombinant IspD from S. coelicolor which
had fairly low affinity for both substrates (Cane et al., 2001).
Cane et al. also reported the native IspD from the crude, cell-
free S. coelicolor extract (Cane et al., 2001). The recombinant
MtIspD had almost the same Km for CTP with native IspD
from S. coelicolor, while Km for MEP is 100-folded lower
than both native and recombinant IspD from S. coelicolor.
This enzymatic property is contrast to their closer phylogenetic
relationship, possibly as a result of their structural variation in
the substrate binding sites.
The recombinant MtIspD had specificity for nucleotide
triphosphates. GTP, UTP, and ITP could not be used as
substrates; only 13% of activity was observed when CTP was
replaced by ATP (data not shown). According to structure of
EcIspD, this selectivity for the pyrimidine base is achieved
through hydrogen bonding interactions of the glycine-rich
loop and steric constrictions in the base-binding pocket that do
not allow for the sequestration of larger purine bases (Richard
et al., 2001). The divalent cation requirement was also
evaluated (Fig. 5C). Divalent cations including Mg2+, Mn2+,
Co2+ or Fe2+ support enzyme activities, the maximal activity
was found with Mg2+. The addition of 2 mM EDTA decreases
enzyme activity. Obviously, MtIspD is a metal-dependent
enzyme and Mg2+ is a preferential cofactor. This is in line with
other monofunctional enzymes of EcIspD and AtIspD
(Rohdich et al., 1999; Rohdich et al., 2000). However, the
bifunctional enzyme CjIspDF is different from these. It is
catalytically active in the presence of various divalent cations,
with Zn2+ supporting the maximal activity (Gabrielsen et al.,
The enzyme activity was analyzed in 50 mM Tris-HCl at
pH range of 6.0~9.0. The optimal pH for this reaction is 8.0
(data not shown). The thermal stability of MtIspD was also
investigated. Fig. 5D presented that the recombinant MtIspD
lost enzyme activity sharply after pre-incubation for 10 min
above 50oC, and almost all of activity was abolished at 80oC.
It suggested that MtIspD is thermal instable. This phenomenon
is opposite to the stability of MtIspD at low temperature. The
purified MtIspD is stable to freezing and maintained its
activity for at least one month when stored at 4oC and for
several months at −80oC.
Table 1. Purification of the recombinant MtIspD
StepsTotal protein (mg)Total activity(U)b
Specific acticity (U/mg)Yield (%)
aThe supernatant proteins was from 1 l culture medium.
bOne unit of enzyme activity (U) is defined as the amount of enzyme that converse 1 µmol substrate per minute at 30oC.
Table 2. Comparison kinetic parameters of MtIspD with homologous IspD proteins
(µmol · min−1· mg−1)
Kcat (s−1)KmMEP (µM)KmCTP (µM)References
M. tuberculosis (MtIspD)
E. coli (EtIspD)
C. jejuni (CjIspDF)
S. coelicolor (ScIspD)
S. coelicolor (Native)b
A. thaliana (AtIspD)
2 × 10−3
Rohdich et al., 1999
Cane et al., 2001
Gabrielsen et al., 2004b
Cane et al., 2001
Cane et al., 2001
Rohdich et al., 2000
a ND, not determined.
bEnzyme from the crude, cell-free S. coelicolor extract.
cKcat was calculated according the value of Vmax and provided molecular weight in the paper.
Properties of 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase from M. tuberculosis 917
Structural properties of MtIspD. Circular dichroism (CD)
spectroscopy, the most widely used chiroptical method, has an
extreme sensitivity toward protein structure. In order to
examine whether conformational changes are responsible for
thermal instability of the recombinant MtIspD, we determined
the secondary and tertiary structures of MtIspD at the
temperature range of 25-80oC by the far-UV and near-UV CD
spectra respectively (Fig. 6).
For the far-UV spectra, MtIspD exhibited similar two negative
extrema in the vicinity of 208 and 222 nm, one positive
extremum at 193 nm, which are characteristic spectra of α + β
or α/β proteins (Venyaminov et al. 1994). Analysis of CD
spectrum at 25oC indicated that the secondary structures for α-
helix, β-sheet, β-turn and random coil were 33.4, 26.6, 11.0
and 29.0%, respectively. Generally, CD spectrum provides
more precise estimates for á-helix (Baumruk et al. 1996). This
α-helix percentage is consistent with predictions of 33.8% by
GOR 4 and 36.4% by JPRED software. It also matches to
33.2% α-helix derived from x-ray analysis of EcIspD three-
dimensional structure (Richard et al. 2001). Therefore, it is
indicative of the native-folded structures of the recombinant
As increments of temperatures, MtIspD displays similar
pattern of spectra in far-UV region, only with minor changes
in the proximity to 193nm and 222nm, revealing that MtIspD
still partly keeps the integrity of secondary structure at higher
temperature from 50oC to 80oC In near-UV region, MtIspD
displays similar shapes at 25, 30 and 40oC, indicative of well-
Fig. 3. Purification of MtIspD. (A) SDS-PAGE assay of the purified MtIspD protein. lane 1, molecular mass marker; lane 2, purified
recombinant MtIspD; lane 3, boiled E. coli cells containing pET28a-MtispD after induction period with 1 mM IPTG. (B) Mass spectrum
analysis of MtIspD.
Fig. 4. Estimation of the molecular weight of MtIspD. (A) MtIspD and standard proteins were submitted to polyacrylamide gel
electrophoresis under nondenaturing conditions in gels of different polyacrylamide concentration. A nondenaturing electrophoresis gel with
7% polyacrylamide was presented. (1) α-lactalbumin (14.2 kDa), (2) carbonic anhydrase (29 kDa), (3) chicken egg albumin (45 kDa), (4)
bovine serum albumin (monomer, 66 kDa, and dimer, 132 kDa), (5) MtIspD. (B) A plot of the negative slopes versus the known molecular
weights of the standards, from which the molecular weight of MtIspD was determined.
918Wenjun Shi et al.
Fig. 5. Enzymatic properties of MtIspD. (A) Effects of MEP concentrations on MtIspD. CTP was held at 2 mM, with varied concentrations
of MEP. (B) Effects of CTP concentrations on MtIspD. MEP was held at 2 mM, with varied concentrations of CTP. (C) Divalent cation
requirements of MtIspD. Divalent cations and EDTA were added to the reaction buffer at a final concentration of 2 mM. (D) Effect of
temperatures on MtIspD enzyme activity. The proteins were pre-incubated at various temperatures ranging from 30oC to 80oC for 10 min and
chilled on ice. The residual activity was measured at 30oC in the standard kinetic assay system, and was expressed as a percentage of the
original activity without preincubation.
Fig. 6. CD spectra of MtIspD at the temperature range of 25-80oC. (A) Far-UV CD spectra of MtIspD were recorded using 0.1 cm path-
length cell. The protein concentration was 0.2 mg/ml in 5 mM Tris-HCl pH 8.0. (B) Near-UV CD spectra of MtIspD were recorded using
1 cm path-length cell. The protein concentration was 2 mg/ml in 5 mM Tris-HCl pH 8.0. The color code is: black, 25oC; red, 30oC; blue,
40oC; green, 50oC; magenta, 60oC; yellow, 70oC; purple 80oC.
Properties of 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase from M. tuberculosis919
defined, native-like tertiary structures. Upon a further increase
of temperature from 50oC to 80oC, less ellipticities were
observed in the 260-285 nm region, suggesting the occurrence
of a thermally-induced unfolding from 50oC to 80oC. This
three-dimensional conformation change above 50oC is consistent
to enzymatic result. Thus, conformational alteration is closely
related with catalytic activity loss. Since the enzymatic active
site of IspD is located at the dimer interface formed by partner
subunits, this thermal instability of MtIspD may possibly
reflect to conformational alteration of the active site influenced
by the tertiary conformational change of each subunit.
In this paper, we first described expression, purification and
characterization of IspD protein from M. tuberculosis H37Rv.
Our results indicated that the recombinant MtIspD is a right-
folded and functionally active homodimer protein. It shares
some basic characteristics with other bacterial monofunctional
IspD proteins. Still, MtIspD bears some unique features.
Substrate affinities of MtIspD are distinct from its phylogenetic
relationship with S. coelicolor. MtIspD is thermal instable above
50oC. As MtIspD is a promising target for anti-tuberculosis
drug design, the biophysical and biochemical is expected to
favor better understanding MtIspD features. Therefore, our
study might gain insight into structural and functional features
of MtIspD and further providing possible hints in the
discovery of the anti-tuberculosis compounds using MtIspD
as a target.
of Science and Technology of China. (973 Project, No.
2002CB512804 and 30670109) and Shanghai Basic Research
Project from the Shanghai Science and Technology Commission
(Grant No. 06JC14012). We are grateful Dr Xiao-ping Chen
and Dr Ya-lei Dai for critically reviewing the manuscript.
The work was supported by the Ministry
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