An a-proteobacterial type malate dehydrogenase may complement
LDH function in
Plasmodium falciparum
Cloning and biochemical characterization of the enzyme
Abhai K. Tripathi
1
, Prashant V. Desai
2
, Anupam Pradhan
1
, Shabana I. Khan
1
, Mitchell A. Avery
1,2
,
Larry A. Walker
1,3
and Babu L. Tekwani
1
1
National Center for Natural Product Research, Research Institute of Pharmacological Sciences,
2
Department of Medicinal
Chemistry, and
3
Department of Pharmacology, School of Pharmacy, University of Mississippi, MS, USA
Malate dehydrogenase (MDH) may be important in car-
bohydrate and energy metabolism in malarial parasites. The
cDNA corresponding to the MDH gene, identified on
chromosome 6 of the Plasmodium falciparum genome, was
amplified by RT-PCR, cloned and overexpressed in
Escherichia coli. The recombinant Pf MDH was purified
to homogeneity and biochemically characterized as an
NAD
+
(H)-specific MDH, which catalysed reversible inter-
conversion of malate to oxaloacetate. Pf MDH could not
use NADP/NADPH as a cofactor, but used acetylpyridine
adenine dinucleoide, an analogue of NAD. The enzyme
exhibited strict substrate and cofactor specificity. The highest
levels of Pf MDH transcripts were detected in trophozoites
while the Pf MDH protein level remained high in troph-
ozoites as well as schizonts. A highly refined model of
Pf MDH revealed distinct structural characteristics of sub-
strate and cofactor binding sites and important amino acid
residues lining these pockets. The active site amino acid
residues involved in substrate binding were conserved in
Pf MDH but the N-terminal glycine motif, which is involved
in nucleotide binding, was similar to the GXGXXG signa-
ture sequence found in Pf LDH and also in a-proteobacterial
MDHs. Oxamic acid did not inhibit Pf MDH, while gossy-
pol, which interacts at the nucleotide binding site of oxido-
reductases and shows antimalarial activity, inhibited
Pf MDH also. Treatment of a synchronized culture of
P. falciparum trophozoites with gossypol caused induction
in expression of Pf MDH, while expression of Pf LDH was
reduced and expression of malate:quinone oxidoreductase
remained unchanged. Pf MDH may complement Pf LDH
function of NAD/NADH coupling in malaria parasites.
Thus, dual inhibitors of Pf MDH and Pf LDH may be
required to target this pathway and to develop potential new
antimalarial drugs.
Keywords: gossypol; lactate dehydrogenase; malate dehy-
drogenase; malate:quinone oxidoreductase; Plasmodium
falciparum.
The enzymes associated with carbohydrate and energy
metabolism in malarial parasites have attracted significant
attention as potential targets for new antimalarial drug
discovery [1]. During asexual reproduction and growth
within the host’s erythrocytes the parasite depends mainly on
the glycolytic pathway to obtain energy. Infected erythro-
cytes consume almost 100 times more glucose than unin-
fected erythrocytes [2]. Almost all of this increase in glucose
utilization is the result of the synthesis of enzymes of
glycolytic (and associated) pathways by the parasite.
Fulminating malaria infections are characterized by hypo-
glycemia and potentially lethal lactic acidosis [3]. Earlier
studies and the recent release of a fully annotated map of the
Plasmodium falciparum genome have shown the presence of
a complete battery of enzymes of the Embden–Meyerhof–
Parnas pathway of glycolysis and the tricarboxylic acid
(TCA) cycle in malarial parasites [4]. However, the role of
oxidative metabolism in the malaria parasite through the
TCA cycle remains unclear. A recent report has indicated the
operation of oxidative phosphorylation and the presence of
an alternative NADH-Q oxidoreductase and malate:qui-
none oxidoreductase in Plasmodium yoelii [5]. The oxidative
metabolism in malaria parasite may be important for de novo
pyrimidine biosynthesis rather than energy metabolism [1].
L
-Malate dehydrogenase (MDH; EC 1.1.1.37) and
L
-lactate dehydrogenase (LDH; EC 1.1.1.27) are 2-keto-
acid:NAD(P) oxidoreductases that are universally distri-
buted in both eukaryotic and prokaryotic organisms [6,7].
LDH is particularly important in anaerobic metabolism,
Correspondence to B. L. Tekwani, National Center for Natural
Product Research, School of Pharmacy, University of Mississippi,
MS 38677, USA. Fax: +1 662 915 7062, Tel.: +1 662 915 7882,
E-mail: btekwani@olemiss.edu
Abbreviations: IPTG, isopropyl thio-b-
D
-galactoside; Pf MDH, Plas-
modium falciparum malate dehydrogenase; Pf LDH, P. falciparum
lactate dehydrogenase; Pf MQO, P. falciparum malate : quinone
oxidoreductase; OAA, oxaloacetate; APAD, acetyl pyridine adenine
dinucleotide; RBC, red blood cells; TCA, tricarboxylic acid.
Enzymes:
L
-Malate dehydrogenase (MDH; EC 1.1.1.37);
L
-lactate
dehydrogenase (LDH; EC 1.1.1.27).
Notes: Part of the work reported in this paper was presented at the 51st
Annual Meeting of American Society of Tropical Medicine & Hygiene,
held at Denver, November 10–14, 2002 [Am. J. Trop. Med. Hyg.
(2002) 67, 146]. The cDNA sequence reported in paper has been
submitted to GenBank under accession no AY324107.
(Received 2 March 2004, revised 2 June 2004, accepted 7 July 2004)
Eur. J. Biochem. 271, 3488–3502 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04281.x
while MDH may have a role in oxidative metabolism as well
as other physiological functions, depending on its biochemi-
cal characteristics and intracellular localization [8]. LDHs
are cytosolic proteins but isoforms of MDH have been
localized to the cytosol as well as different subcellular
organelles such as mitochondria, chloroplast, peroxysomes
and glyoxysomes [8]. LDHs and MDHs characterized from
different organisms form a large superfamily [9]. A specific
phylogenetic distribution of LDH, LDH-like MDH and
dimeric MDH over the Archaeal, Bacterial and Eukaryal
domains was observed. All LDHs and MDHs enzymes
from apicomplexan parasites, which include Plasmodium
spp., Toxoplasma gondii,Cryptosporidium parvum and
Eimeria tenella, were found to be monophylectic within
the ÔLDH-like MDHÕgroup as a sister to alpha-proteobac-
terial MDHs [10]. All of the apicomplexan LDHs, with the
exception of LDH1 from Cryptosporidium parvum,forma
separate clade from their MDH counterparts, indicating
that these LDHs evolved from an ancestral apicomplexan
MDH by gene duplication and functional conversion before
expansion of apicomplexans [10,11]. Both LDH and MDH
from various organisms have been characterized in consid-
erable detail at the molecular and structural levels
[6,7,12,13]. However, only LDH from P. falciparum
(Pf LDH) has been characterized in significant detail,
including determination of its crystal structure. The enzyme
has also been exploited as a potential target for design and
development of specific enzyme inhibitors as antimalarial
agents [14–17]. A comparative kinetic and structural
analysis of LDH from all four species of human malaria
parasite has also been reported recently [18]. However,
MDH, which catalyses reversible conversion of malate to
oxaloacetate (OAA) using NAD(P) as a cofactor, has not
been characterized in sufficient detail from P. falciparum.It
was shown previously that P. falciparum contained a single
isozyme of MDH (Pf MDH) which was suggested to be
localized to the cytosol of the parasite [19]. The avian
malaria parasite P. lophurae was also found to contain a
single isozyme of MDH which was distinct from host
erythrocyte MDH [20]. Alternatively, P. berghei the rodent
malaria parasite has been shown to have multiple MDH
isozymes [21]. However, the presence of multiple MDH
isoforms in mouse erythrocytes, the preference of P. berghei
to infect reticulocytes, and the detection of low activity of
MDH in P. berghei raises questions regarding the validity of
this report. The recent release of the P. falciparum genome
sequence has indicated the presence of a gene putatively
encoding for MDH [4]. Initial analysis of the Pf MDH
sequence revealed its higher homology with the MDH
sequences from a-proteobacteria and also with LDHs.
Pf MDH may therefore be classified as LDH-like MDH,
similar to that from other apicomplexan parasites [10,11].
In this study Pf MDH was cloned from P. falciparum
genomic DNA and total RNA by PCR and RT-PCR,
overexpressed in Escherichia coli and the recombinant
enzyme was purified to homogeneity. Functional charac-
terization of the recombinant Pf MDH protein was carried
out by analysis of its biochemical characteristics and enzyme
kinetics. Expression of MDH in P. falciparum was found to
be developmentally regulated during its growth and prolif-
eration in human erythrocytes. A highly refined homology
model for Pf MDH was also constructed on the basis of
crystal structures of nearest structural homologues i.e.
Pf LDH [14], E. coli MDH [22] and Chlorobium tepidum
MDH [23]. High homology of Pf MDH with Pf LDH also
prompted us to ask whether MDH can complement the
function of LDH in the malaria parasite. This was studied
by treating the P. falciparum cultures with gossypol, an
inhibitor of LDH, which has also shown antimalarial
action [24].
Materials and methods
P. falciparum (D6 strain) was grown in vitro in RPMI 1640
medium with A+ human red blood cells (RBCs) and A+
human serum as described previously [25]. For large-scale
culture the parasite was grown in 75 cm
2
culture flasks,
which can accommodate up to 200 mL of culture medium.
The parasite was grown in 24 culture flasks to 10–15%
parasitemia. The cultures were highly synchronized with
two/three cycles of sorbitol treatment [26]. The parasite
cultures initiated with early ring stage were harvested at
regular 8 h intervals starting from 0 to 40 h to isolate the
parasite at different developmental stages of life cycle viz.,
early rings, late rings, early trophozoites, late trophozoites,
early schizonts and late/mature schizonts. The RBC-free
parasite was prepared by lysis of infected RBCs with
saponin [27]. Genomic DNA and RNA were prepared by
using a genomic DNA isolation kit (Qiagen) and trizol
method (Invitrogen), respectively, as per the manufacturer’s
protocol.
Cloning of
Pf
MDH,
Pf
LDH and
Pf
MQO
Pf MDH was cloned for functional and biochemical
characterization. The other two genes Pf LDH and
Pf MQO were also cloned to obtain the DNA probes for
analysis of its expression in P. falciparum cultures. Pf LDH
was also overexpressed in E. coli cultures to obtain the
enzyme for evaluation of the inhibitors. The primers
(forward primer 5¢-ACTAAAATTGCTTTAATAGG
TAG-3¢and reverse primer 5¢-TTATTTAATGTC
GAAAGC-3¢) for cloning of full-length MDH ORF
DNA and cDNA corresponding to the complete coding
sequence were designed from the putative MDH gene
(MAL6P1.242) identified on chromosome 6 of P. falcipa-
rum (http://www.plasmodb.org). The DNA corresponding
to full-length Pf MDH ORF was amplified by PCR using
Taq DNA polymerase and P. falciparum genomic DNA as
the template. The cDNA was prepared by RT-PCR using
DNase-treated total RNA, isolated from mixed culture of
P. falciparum as the template using a Qiagen RT-PCR kit.
The control reaction without reverse transcriptase was run
simultaneously to check for genomic DNA contamination
and to ensure specific amplification of cDNA. The DNA
corresponding to full-length ORF sequence of Pf LDH
(PF13-0141) and Pf MQO (MAL6P1.258) were also ampli-
fied using the appropriate primers (Pf LDH: forward
5¢-ATGGCACCAAAAGCAAAAATCG-3¢and reverse
5¢-AGCTAATGCCTTCATTCTCTTAG-3¢;Pf MQO for-
ward 5¢-ATGATATGTGTTAAAAATATTTTG-3¢and
reverse 5¢-TCATAAATAATTAACGGGATATTCG-3¢).
Both PCR and RT-PCR products were cloned directly into
an E. coli expression vector (pQE30) using a UA cloning kit
FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3489
(Qiagen). The ligation mixtures were used to transform
EcoliXL-1 blue cells and the bacterial colonies transformed
with recombinant plasmids were selected on Luria–Bertani
medium agar plates containing 100 lgÆmL
)1
ampicillin. The
presence of the DNA/cDNA inserts, and their orientation in
the recombinant plasmids, was confirmed by digestion of
the plasmid minipreps with appropriate restriction enzymes
and their analysis by electrophoresis on 0.8% (w/v) agarose
gels. The plasmid containing Pf MDH ORF (DNA and
cDNA) and Pf MQO cDNA in the correct orientation were
sent to Laragen Inc. (http://www.laragen.com) for sequen-
cing on both strands. The Pf MDH mRNA sequence for
P. falciparum (Sierra Leone D6 strain) has been submitted
to GenBank under accession no AAQ23154 and was found
to be the same as that reported for a putative MDH
sequence of a 3D7 P. falciparum strain. The recombinant
pQE30Pf LDH and pQE30Pf MQO plasmids were also
analysed in the same way.
Overexpression and purification of recombinant proteins
For overexpression of Pf MDH, Pf LDH and Pf MQO
the recombinant plasmids prepared from XL-1 blue cells
were transformed into E. coli M15 (pREP 4) cells as
recommended by Qiagen. The transformed colonies were
selected on LB agar plates containing 100 lgÆmL
)1
ampicillin and 25 lgÆmL
)1
kanamycin. A 5 mL overnight
culture of transformed E. coli cells was transferred to
500 mL fresh LB medium containing ampicillin and
kanamycin and grown further at 37 CtoD
600
0.5. At
this time, isopropyl thio-b-
D
-galactoside (IPTG) was
added to the cultures to a final concentration of
0.5 m
M
in order to induce overexpression of recombinant
proteins. The cultures were grown for additional 5 h at
37 C with constant shaking. Cells were harvested by
centrifugation at 5000 gfor 15 min at 4 C. Harvested
bacterial pellets were re-suspended in phosphate buffer
containing 300 m
M
NaCl and 10 m
M
imidazole and lysed
by sonication. The extracts were centrifuged at 4 Cat
15 000 gfor 30 min. Expression of recombinant protein
was checked by SDS/PAGE and also by Western
blotting. Significant overexpression of Pf MDH and
Pf LDH was achieved in E. coli cultures induced with
IPTG. Expression of recombinant Pf MDH and Pf LDH
could be achieved in soluble fractions. However, over-
expression of Pf MQO could not be achieved in this
system, even with the use of various concentrations of
IPTG and varying culture conditions. The soluble frac-
tions, which contained significant amounts of the
Pf MDH/Pf LDH protein, were used for purification.
The recombinant proteins contain a 6 ·His tag which
facilitates their purification by affinity chromatography
using Ni–NTA chelating columns. The soluble bacterial
extracts were passed through Ni–NTA agarose columns
and the columns were washed with buffer containing
20 m
M
imidazole. Recombinant proteins were eluted with
buffer containing 200 m
M
imidazole. The soluble extracts,
column fractions and purified proteins were analysed by
SDS/PAGE. Gels were stained with Coomassie brilliant
blue R and also with silver stain, to check the purity of
recombinant proteins. The oligomeric structure of the en-
zymatically functional recombinant Pf MDH preparations
was determined by a standard size exclusion chromatog-
raphy procedure using Sephacryl S200.
Enzyme assays
MDH catalyses reversible oxidation of malate to OAA
utilizing NAD(H) or NADP(H) as cofactor. MDH was
assayed spectrophotometrically for both oxidation of
malate and reduction of OAA by recording the change
in absorbance at 340 nm as described earlier [19]. The
assays were set up in clear flat-bottomed 96-well micro-
plates. For the malate reduction assay, the reaction
mixture contained in a total volume of 200 lLwith
glycine buffer (pH 10.2, 50 m
M
) malate (20 m
M
or as
specified) and NAD or NADP (500 l
M
or as specified)
and an appropriate amount of the enzyme. Similarly for
the OAA reduction assay the reaction mixture contained
in a total volume of 200 lL with phosphate buffer
(pH 7.0, 50 m
M
), OAA (250 l
M
or as specified) and
NADH or NADPH (200 l
M
or as specified). Appropriate
controls without substrates or cofactor were also set up
simultaneously. Each assay was set up in triplicate. The
plates were read on a microplate reader in kinetic mode
for 5 min and the change in absorbance per minute was
recorded. Activity of the enzyme was calculated in terms
of micromoles of NAD reduced or NADH oxidized.
Similarly Pf LDH activity was also measured according to
the spectrophotometric method as described earlier [16].
For determination of optimum pH for pfMDH activity
the assays were performed at different pH ranging from
6.0 to 11.0 using phosphate (pH 6.0–8.0) and glycine
(pH 8.5–11.0) buffers with saturating concentrations of
the substrate and the cofactor. For enzyme kinetic
studies, assays were performed at varying concentrations
of substrates (OAA or malate) and the cofactors [NAD/
H or acetyl pyridine adenine dinucleotide (APAD/H)].
Kinetic constants were computed with
GRAFIT
5. To
determine substrate specificity of Pf MDH the enzyme
activity was also determined using different 2-keto or
2-hydroxy carboxylic acids such as lactate, pyruvate,
a-ketoglutarate, keto-malonate, oxo-butyrate and keto-
adipic acid.
Analysis of expression of the enzymes in
P. falciparum
cultures
Expression of Pf MDH at the different stages of the asexual
intra-erythrocytic cycle was evaluated by semiquantitative
analysis of transcripts by Northern blotting and also by
quantification of the enzyme protein by Western blotting.
Expression of Pf MDH, Pf LDH and Pf MQO in control
and drug treated P. falciparum cultures was evaluated by
Northern and Western blotting. Quantitative analysis of the
transcripts and proteins was performed by using the NIH
IMAGE
(version 1.61) analysis program.
Northern blotting. The P. falciparum cultures at different
stages of life cycle or the control and drug treated cultures
were harvested by centrifugation. Total RNA was isolated
directly from the infected RBCs by the Trizolmethod as
per the manufacturer’s protocol. Purity and concentration
of RNA was checked spectrophotometrically by reading the
3490 A. K. Tripathi et al. (Eur. J. Biochem. 271)FEBS 2004
absorbance at 260/280 nm. An equal amount of total RNA
(20 lg) from each of the six stages or untreated and drug
treated samples was separated on denaturing 1% (w/v)
agarose gel. To check the concentration and purity of RNA
the gels were stained with ethidium bromide and visualized
on a UV transluminator. Equal intensity of ribosomal RNA
bands in all of the lanes indicated that an equal amount of
total RNA had been loaded. The RNA from agarose gels
was transferred under vacuum to positively charged nylon
membrane (Sigma) and the membranes were washed with
0.1% NaCl/Cit and 0.1% SDS for 30 min and prehybrid-
ized in 10 mL prehybridization solution [3.6 mL sonicated
salmon sperm DNA of a 10 mgÆmL
)1
stock, 0.2 mL 50·
Denhardt’s solution, 0.1 mL 10% (w/v) SDS, 3.64 mL
10 mgÆmL
)1
and 3 mL 20·NaCl/Cit] for 5 h at 42 Cwith
gentle shaking. Membranes were then hybridized with
hybridization solution containing denatured radioactive
Pf MDH or Pf LDH or Pf MQO probe. The recombinant
plasmids containing Pf MDH/Pf LDH/Pf MQO cDNA
inserts were digested with appropriate restriction enzymes
and the inserts were purified on the agarose gel, using a gel
extraction kit (Qiagen). The probes were prepared by
labelling the inserts with [
32
P]dCTP using a random prime
labelling kit (Amersham Biosciences). Hybridization was
performed overnight at 42 C with gentle shaking. The
membranes were then washed successively with 10·NaCl/
Cit, 1% (w/v) SDS (20 min), 1·NaCl/Cit, 0.5% (w/v) SDS
(30 min) and 0.1·NaCl/Cit, 0.2% (w/v) SDS (45 min). The
membranes were exposed to hyperfilm and the transcripts
were visualized by autoradiography.
Western blotting. For quantification of enzyme protein
the parasites were isolated from infected RBCs by
saponin lysis [27]. The parasite pellets were washed
extensively with cold NaCl/P
i
to remove RBC proteins
and membrane contaminants. Finally, the parasite pellet
was resuspended in NaCl/P
i
containing 0.1% (v/v) Triton
X-100 and the proteins were solublized by sonication. The
soluble protein extracts were obtained after centrifugation
of the lysates at 10 000 gfor 15 min in an Eppendorf
microcentrifuge tube. Protein concentrations in the clear
supernatants were determined by the Bradford method
[28]. Equal amount of total protein (100 lg) from each
sample was loaded and separated by SDS/PAGE. Pre-
stained molecular mass protein markers (Sigma-Aldrich)
were also loaded in the first lane. The proteins were
transferred onto nitrocellulose membrane (Sigma) using
semi-dry blot and membrane was probed with purified
IgG fraction obtained from polyclonal antiserum raised
against recombinant Pf MDH (Antibodies Inc.). Pf MDH
protein was visualized using peroxidase conjugated sec-
ondary antibody (anti-rabbit IgG).
To evaluate the effect of gossypol on expression of the
enzymes, highly synchronized cultures of P. falciparum with
10–15% parasitaemia were treated at early trophozoite
stages with gossypol at a concentration of 25 or
100 lgÆmL
)1
in the culture medium. The cultures were
harvested after 8 h and total RNA was isolated from the
infected RBCs and analysed for expression of Pf MDH,
Pf LDH and Pf MQO by Northern blotting as described
above. Parasite was isolated from control and treated
cultures and equal amount of protein was analysed by
Western blotting as described above. Antimalarial activities
of oxamic acid and gossypol were determined in vitro for
P. falciparum cultures as described earlier [29].
Analysis of
Pf
MDH sequence
Protein sequences of various MDHs and LDHs were
obtained from GenBank. All of the selected sequences along
with Pf MDH sequence were aligned using
CLUSTAL
–
WINDOWS
interface with default parameters [30]. Misaligned
or poorly aligned sequences were selected manually and
realigned to obtain proper alignment of all sequences.
PHYLIP
format tree output was selected to obtain the
bootstrap neighbor-joining tree. An unrooted phylogenetic
tree was drawn using
TREE VIEW
. The accession number of
the sequences used for the phylogenetic analysis were:
P. falciparum MDH (NP_703844), P. falciparum LDH
(CAD52397), P. yoelii MDH (EAA22943), P. yoelii LDH
(EAA15666), Ricketssia prowozekii MDH (NP_220759),
Mesorhizobium loti MDH (NP_105210), Sinorhizobium
meliloti MDH (AAG41996), Rhizobium leguminosarum
MDH (CAA05717), Trypanosoma brucei MDH
(AAK83037), human cytoplasmic MDH (P40925), mouse
cytoplasmic MDH (P14152), maize cytoplasmic MDH
(T02935), Arabidopsis thaliana MDH (AAM65532), Tricho-
monas vaginalis MDH (2208292 A), T. vaginalis LDH
(AAC72735), E. coli MDH (AAC76268), E. coli LDH
(NP_418062), C. tepidum MDH (NP_662392), Leishmania
major MDH (CAB55506), S. meliloti LDH (CAC49543),
M. loti LDH (NP_107321), human LDH (P07864), maize
LDH (P29038), and A. thaliana LDH (AAM64829).
Generation of a model structure of
Pf
MDH
Computational studies were performed on a Silicon Graph-
ics Octane 2 workstation, equipped with two parallel
R12000 processors, V6 graphics board and 512 MB mem-
ory. Comparative protein structure modelling was per-
formed with the
HOMOLOGY
module of
INSIGHTII
2000
(Accelrys Inc., San Diego, CA, USA). Energy minimiza-
tions and molecular dynamics were accomplished in the
DISCOVER
module of
INSIGHTII
2000. The geometric and
local environmental consistency of the model was evaluated
with the
PROSTAT
and
PROFILES
-3
D
[31] modules of
INSIGHTII
2000 as well as the
MATCHMAKER
[32] module of
SYBYL
6.9
(Tripos Associates Inc., St. Louis, MO, USA).
A
WU
-
BLAST
2.0 [33] PDB search was performed on the
Pf MDH sequence with default parameters of
BLASTP
gapped alignment. Only two sequences, MDH from
C. tepidum (40% sequence identity) and Pf LDH (39%
sequence identity) passed the identity filter of 35% and
hence were used to build the 3D model. A total of nine
structurally conserved regions and seven structurally
variable regions or loops were identified. The structurally
conserved regions were built from the homologues
whereas the coordinates for the loops were obtained by
searching the PDB for regions of proteins that meet a
defined geometric criterion. The protocol uses an existing
Cacarbon distance matrix to search for regions of
proteins whose Cadistances best fit those of the selected
region of the protein being studied, while meeting the
additional constraint of having the specified number of
FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3491
residues present between the regions of interest. Different
rotamers for the residues that line the active pocket were
also studied and the most energy stable rotamers were
retained. The crude model so obtained was then refined
by minimization using an analogous approach reported
previously [34].
The structure of the Pf MDH in complex with OAA and
NADH was obtained as follows. First, the substrate and the
cofactor were placed at appropriate binding regions of
Pf MDH based on their corresponding locations in the
Pf LDH [14] and C. tepidum MDH structures [23]. The
complex was then minimized using steepest descents for
1000 iterations followed by 2000 iterations of conjugate
gradients. This was followed by molecular dynamics
simulations for 25 ps at 300 K and finally minimization to
a gradient of 0.001 kcalÆmol
)1
ÆA
˚
)1
or less using conjugate
gradients. During this refinement, the side chains of the
residues lining the substrate as well as the cofactor binding
pockets were allowed to move freely whereas rest of the
protein atoms were fixed. Comparison of the Pf MDH
model structure with crystal structures of Pf LDH [14],
C. tepidum MDH [23], E. coli MDH [35] and T. gondii
LDH [36] was achieved by determination of the rmsd
between each pair. This is one of the most acceptable
methods for determination of structural similarity among
the proteins [37].
Results
Cloning and characterization of
Pf
MDH mRNA and gene
Preparation and amplification of cDNA corresponding to
the complete encoding region of Pf MDH by RT-PCR
yielded a single amplicon of 942 bases, which encodes a
predicted protein of 313 amino acids with calculated
molecular mass of 34 040 Da. The sequence of Pf MDH
mRNA from P. falciparum (Sierra Leone D6 strain) was
found to be the same as reported for the Pf MDH gene
identified on chromosome 6 of P. falciparum (3D7 strain).
The results confirm that the PfMDH gene had no introns.
Presence of Pf MDH mRNA in P. falciparum cultures also
indicated that the parasite expressed the gene during the
asexual reproduction cycle. The sequence of the Pf MDH
protein was subjected to
BLAST
-
P
analysis with non-
redundant GenBank protein database. Comparison of
Pf MDH sequence with some representative MDH
sequences are presented in Fig. 1. The sequence of Pf LDH
was also included in this as Pf MDH showed significant
similarity with a-proteobacterial MDHs and also with
LDHs characterized in bacteria and lower eukaryotes,
particularly that from apicomplexan parasites. Pf MDH
did not possess any target signal sequence and therefore
represents a cytosolic MDH. This was further confirmed
by the observation that Pf MDH sequence shows higher
homology with cysolic MDH than with organellar MDHs
(Fig. 2). The Pf MDH protein sequence also contained
several conserved motifs and amino acids residues that
have been found to be important in MDH and LDH
functions (Fig. 1). The N-terminal glycine motif which is
involved in nucleotide binding function was identified as
GSGQIG in Pf MDH and corresponded to the signature
sequence GXGXXG, found in proteobacterial MDHs and
LDHs, rather than the signature sequence of GXXGXXG
found in most of the MDHs. More detailed analysis of
substrate and nucleotide binding sites has been described in
the section on structural analysis of Pf MDH by homology
modelling.
Overexpression and biochemical characterization
of
Pf
MDH
Overexpression of Pf MDH cDNA in E. coli yielded a
recombinant protein of 36 kDa as analysed by SDS/
PAGE (Fig. 3). The recombinant protein expressed in
E. coli was larger then the predicted Pf MDH protein of
34 kDa because it also contained some extra amino acids
derived from the bacterial expression vector, including a
His
6
-tag at the N-terminal end, which facilitates its purifi-
cation. Most of the recombinant protein was recovered in
the soluble fraction and was found to be functionally active
as MDH, i.e. it catalysed reduction of OAA and oxidation
of malate in the presence of NADH/NAD. The protein
could be purified to apparent homogeneity, as analysed by
SDS/PAGE, after single-step affinity chromatography
through a Ni–NTA agarose column. About 10 mg of the
functionally active recombinant Pf MDH could be recov-
ered from 1 L of the E. coli culture.
The homogeneous preparations of recombinant Pf MDH
were used to characterize oligomeric status, enzyme kinetic
properties, and substrate/cofactor specificities. Analysis of
recombinant Pf MDH by size exclusion chromatography
on Sepahcryl S200 did not provide conclusive information
on the oligomeric state of the enzymatically active protein.
Both dimeric as well as terameric forms of the proteins were
eluted. Pf MDHmayexistasadimerofdimers.MDH
catalyses interconversion of malate and OAA inside the cell.
However, in vitro these reactions occur at a different pH.
Therefore, before a detailed kinetic analysis of the enzyme
could be conducted, the optimum pH for oxidation of
malate and reduction of OAA were determined by per-
forming the assays at varying pH (5.5–11.0) using different
reaction buffers (Fig. 4). The optimum pH for oxidation of
malate in the presence of NAD was 10.2 and for the
reduction of OAA using NADH as the cofactor it was 7.0.
Further kinetic characterization of enzyme was performed
at the experimentally determined pH optima. Pf MDH
catalysed the reduction of OAA with an efficiency six to
eight times greater than that of oxidation of malate as
determined by evaluation of V
max
and k
cat
values for
malate/NAD and OAA/NADH (Table 1). The saturating
concentrations of OAA and NADH were found to be
250 l
M
and 150 l
M
, respectively, while for malate and
NAD these were 20 m
M
and 1.5 m
M
, respectively.
Further increase in the concentration of the substrates or
cofactor did not cause any inhibition in enzyme activity.
Importantly Pf MDH did not show characteristic substrate
inhibition. Pf MDH did not utilize NADP/NADPH as an
alternate cofactor, having been tested up to maximum
concentration of 100 m
M
. A surprising observation was the
use of acetyl pyridine adenine dinucleotide (APAD/
APADH) as an alternate cofactor by Pf MDH, which
showed almost comparable efficiency to that of NAD/
NADH (Table 1). Porcine heart MDH (M7383, Sigma-
Aldrich) did not show any activity with APAD/APADH
3492 A. K. Tripathi et al. (Eur. J. Biochem. 271)FEBS 2004