Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv.
ABSTRACT 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-methylerythritol (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 Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. 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.
- [Show abstract] [Hide abstract]
ABSTRACT: Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD; EC 126.96.36.199) is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis.European journal of pharmacology 09/2012; 694(1-3):45-52. · 2.59 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: 2-C-methyl-D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME), another key intermediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-Methyl-D-erythritol-4-phosphoric acid from D-(+)-arabitol.Tetrahedron 10/2012; 68(43):8937-8941. · 2.80 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPS) are considered a major virulence factor and are targets for a vaccine. D-mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster were proposed to be responsible for the synthesis of NDP-D-mannitol (the nucleotide activated form of D-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis, and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to D-fructose-6-phosphate to form CDP-D-fructose while Mnp2 catalyzed the conversion of CDP-D-fructose to CDP-D-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as D-fructose-6-phosphate cytidylyltransferase encoding gene, and mnp2 (renamed as mnpB) as a CDP-D-fructose reductase encoding gene. The kinetics of Mnp1 for the substrate (D-fructose-6-phosphate and CTP) and of Mnp2 for the substrate (CDP-D-fructose) and the cofactor (NADH or NADPH) fitted the Michaelis-Menten model. The effects of temperature, pH, and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-D-mannitol has been identified biochemically.Glycobiology 07/2012; · 3.54 Impact Factor
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: email@example.com (H. Wang)
firstname.lastname@example.org (X. Zhang)
912 Wenjun 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 188.8.131.52) 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. tuberculosis 915
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.