Báo cáo khoa học: An a-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum Cloning and biochemical characterization of the enzyme potx

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 a nd overexpressed in

Escherichia coli. The recombinant Pf MDH w as 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 a s a cofactor, but used acetylpyridine
adenine dinucleoide, an analogue of NAD. The enzyme
exhibited s trict substrate and cofactor specificity. The h ighest
levels of Pf MDH transcripts were detected in trophozoites
while the Pf MDH protein level remained high i n troph-
ozoites as well a s 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 b inding were con served in
Pf MDH but the N-terminal glycine motif, which is involved
in nucleotide binding, was similar to the GXGXXG signa-
ture sequence f ound in Pf LDH and also in a-proteobacterial
MDHs. O xamic acid did not inhibit Pf MDH, while gossy-
pol, which interacts at the nucleotid e 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 t arget t his pathway and to develop potential n ew

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 poten tial targets for new a ntimalarial drug
discovery [1]. During asexual reproduction and growth
within the host’s erythrocytes t he 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 i ncrease in glucose
utilization is the result of the synthesis of enzymes of
glycolytic (and a ssociated) p athways by t he parasite.
Fulminating malaria infe ctions are characterized by hypo-
glycemia and potentially lethal lactic acidosis [3]. Earlier
studies and the re cent release of a fully annotated map of the
Plasmodium falciparum genome have shown the presence of
a c omplete battery of enzymes of the Embden–Meyerhof–
Parnas pathway o f 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 i n malaria parasite may be importan t 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.: +1 662 915 788 2,
E-mail:
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 b een
submitted to Ge nBank under accession no AY324107.
(Received 2 March 2004, revised 2 June 2004, accepted 7 July 2004)
Eur. J. Biochem. 271, 3488–3502 (2004) Ó 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., Toxop lasma gondii, Cryptosporidium parvum and
Eimeria tenella, were found to be monophylectic within
the ÔLDH-like MDHÕ group as a sister to alpha-proteobac-
terial MDHs [10]. All of the a picomplexan LDHs, w ith t he
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 c onversion before
expansion of a picomplexans [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 explo ited as a poten tial 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 r eported r ecently [ 18]. Howe ver,

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 s hown previously that P. falciparum contained a single
isozyme of MDH (Pf MDH) which was suggested to be
localized t o t he c ytosol of the parasite [ 19]. T he avia n
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 t he 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 f rom P. falciparum
genomic DNA and total RNA b y PCR and R T-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 enz yme
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 o n the basis of
crystal structures of nearest structural homologues i.e.
Pf LDH [14], E. coli MDH [22] and Chlorobium t epidum
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 goss ypol, an
inhibitor of LDH, which has also shown antimalarial
action [24].
Materials and methods
P. falciparum (D6 s train) was grown in vitro in RPMI 1640
medium with A+ human red b lood 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 c an 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 s orbitol treatment [26]. T he parasite
cultures initiated with e arly ring stage were harvested at
regular 8 h intervals starting from 0 to 40 h t o isolate the
parasite at different developmental stages of life cycle viz.,
early rings, late rings, early trophozoites, late troph ozoites,
early schizonts and late/mature schizonts. The RBC-free
parasite was prepared by lysis of infected RBCs with
saponin [27]. G enomic DNA and RNA were prepared by
using a genomic DNA isolation kit (Qiagen) and trizol
Ò
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 i n 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 c DNA corresponding to the complete coding
sequence were designed from the putative MDH gene
(MAL6P1.242 ) identified on chromosome 6 of P. falcipa-
rum (). 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 MQ O (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
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3489
(Qiagen). The ligation m ixtures were used t o 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 the ir orientation in
the recombinant plasmids, was confirmed by digestion of
the plasmid minipreps with a ppropriate 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. () 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 n o AAQ23154 and was found
to be the s ame as t hat reported f or a putative M DH
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 MD H, Pf LDH a nd Pf MQO
the recombinant plasmids p repared 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 g for 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. T he extracts were centrifuged at 4 °Cat
15 000 g for 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. Expre ssion 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 us e o f v arious con centrations of
IPTG and varying cultur e conditions. T he soluble f rac-
tions, which contained significant amounts of the
Pf MDH/Pf LDH protein, were used for purification.
The recombinant proteins contain a 6 · His t ag which
facilitates their purification by affinity chromatography
using Ni–NTA chelating columns. The s oluble 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 solub le extracts,
column fractions and purified proteins w ere analysed by
SDS/PAGE. Gels were stained with Coomassie brilliant
blue R and also with silver stain, to check t he purity o f
recombinant proteins. The oligomeric structure of the en-
zymatically functional recombinant Pf MDH preparations
was d etermined by a standard size exclusion c hromatog-
raphy procedure u sing 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 a t 340 nm as des cribed earlier [ 19]. T he
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 f or
the OAA reduction assay the reaction mixture c ontained
in a total volume of 20 0 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 c hange 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 a ssays 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 a t 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 l actate, p yruvate,
a-k etoglutarate, 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 t he 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 c entrifugation. Total RNA was isola ted
directly from the infected RBCs by the TrizolÒ method 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) Ó 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 s amples was s eparated 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 t ransluminator. Equal i ntensity of ribosomal RNA
bands in all o f 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 t he 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 u sing a random prime
labelling kit (Amersham Biosciences). Hybridization was
performed overnight at 42 °C with gentle shaking. T he
membranes were t hen 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 w ith cold NaCl/P
i
to r emove 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 t he l ysates at 10 000 g for 15 min in an Eppendorf
microcentrifuge tube. Protein concentrations in the clear
supernatants were d etermined by th e Bradford method
[28]. Equal amount of total protein (100 lg) from each
sample was loaded a nd 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 w ith purified
IgG fraction obtained from polyclonal a ntiserum raised
against recombinant Pf MDH (Antibodies Inc.). Pf MDH
protein w as visualized using peroxidase c onjugated s ec-
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 c ulture medium. The cultures were
harvested after 8 h and total RNA was isolated from the
infected RBCs and analysed f or expression of Pf MDH,
Pf LDH and Pf MQO by N orthern blotting as d escribed
above. Parasite was isolated from control and treated
cultures and equal amount of protein was analysed by
Western blotting as d escribed above. Antimalarial ac tivities
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 o f the selected sequences along
with Pf MD H 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), S inorh izobium
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 t he PDB for regions of proteins that meet a
defined geometric criterion. The protocol uses an existing
Ca carbon distance matrix to search for regions of
proteins whose Ca distances best fit those of the selected
region of the protein being studied, while meeting the
additional constraint of having the specified number of
Ó 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 t he most energy stable rotamers were

retained. The crude m odel so obtained was then refined
by minimization using an a nalogous 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 a t 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 b inding
pockets were allowed to move f reely whereas rest of the
protein a toms were fixed. C omparison o f t he Pf MDH
model s tructure with crystal structures of Pf LDH [14],
C. tepidum MDH [23], E. coli MDH [ 35] a nd T. gondii
LDH [36] was achieved by determination of the rmsd
between each p air. This is one of the most a cceptable
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 c omplete encoding region of Pf MDH by R T-PCR
yielded a single amplicon of 942 bases, which encodes a
predicted protein of 313 amino acids with calculated
molecular mass o f 34 040 Da. T he sequence of Pf MDH
mRNA from P. falciparum (Sierra Leone D6 strain) w as
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 o f Pf MDH mRNA in P. falciparum cultures also
indicated t hat 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 M DH
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 t arget s ignal s equence a nd th erefore
represents a cytosolic MDH. This was further confirmed
by the observation that Pf MDH s equence shows higher
homology with cysolic MDH than with organellar MDHs

(Fig. 2 ). The Pf MDH protein sequence also contained
several c onserved motifs and a mino acids residues that
have been found to be important in MDH and LDH
functions (Fig. 1). The N-terminal g lycine 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 d etailed analysis of
substrate and nucleotide binding sites has been described in
the s ection on structural analysis of Pf MDH by homology
modelling.
Overexpression and biochemical characterization
of
Pf
MDH
Overexpression of Pf MDH c DNA i n E. coli yielded a
recombinant p rotein of  36 kDa a s analysed b y SDS/
PAGE (Fig. 3). The recombinant protein expressed in
E. coli was larger t hen 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 e nd, which facilitate s 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 p resence of N ADH/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 reco mbinant Pf MDH
were used to characterize o ligomeric status, e nzyme 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 b e 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 N AD was 10.2 and for t he
reduction of OAA using NADH as the cofactor i t 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 e fficiency six to
eight times greater than that o f 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 a cetyl 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) Ó FEBS 2004
(results not shown). Pf MDH also showed strict substrate
specificity as no enzyme activity could be detected with
lactate, pyruvate, a-ketoglutarate, oxo-butyrate and
keto-adipic acid. However, a-keto malonate was utilized

by Pf MDH at v ery high concentrations with a K
m
of
5.44 m
M
(Table 1).
P. falciparum(LDH) MAPKAKIVLV-G-SGMIGGVMATLIVQKNLG DVVLFDIVKNMPHGKALD TSHT 51
P. falciparum(MDH) MTKIALI-G-SGQIGAIVGELCLLENLG DLILYDVVPGIPQGKALD LKHF 48
Rickettsia prowazekii MKKNPKISLI-G-SGNIGGTLAHLISLKKLG DIVLFDVSEGLPQGKALD LMQA 51
Rhizobium leguminosarum MARN-KIALI-G-SGMIGGTLAHLAGLKELG DIVLFDIADGIPQGKGLD ISQS 50
Cryptosporidium parvum MR KKISII-G-AGQIGSTIALLLGQKDLG DVYMFDIIEGVPQGKALD LNHC 49
Chlorobium tepidum MKITVI-G-AGNVGATTAFRLAEKQLAR ELVLLDVVEGIPQGKALD MYES 48
E. coli MK-VAVLGAAGGIGQALALLLKTQLPSG-SELSLYDIAPVTP-GVAVD LSH 48
Trypanosoma brucei MSNTCKRVAVTGAAGQIGYSLLPLIAAGRMLGFDQRVQLQLLDISPALKALEGIRAELMD 60
Homo sapiens MSEPIR-VLVTGAAGQIAYSLLYSIGNGSVFGKDQPIILVLLDITPMMGVLDGVLMELQD 59
: * :* :. : : * ::

P. falciparum (LDH) NVMAYSNCKVSGSNTYDDLAGADVVIVTAGFTKAPGKSDKEWNRDDLLPLNNKIMIEIGG 111
P. falciprum (MDH) STILGVNRNILGTNQIEDIKDADIIVITAGVQRKEGMT REDLIGVNGKIMKSVAE 103
Rickettsia provazakii ATIEGSDIKIKGTNDYRDIEGSDAVIITAGLPRKPGMS RDDLISVNTKIMKDVAQ 106
Rhizobium legumunosarum SPVEGFDVNLTGASDYSAIEGADVCIVTAGVARKPGMS RDDLLGINLKVMEQVGA 105
Cryptosporidium parvum MALIGSPAKIFGENNYEYLQNSDVVIITAGVPRKPNMT RSDLLTVNAKIVGSVAE 104
Chlorobium tepidum GPVGLFDTKVTGSNDYADTANSDIVVITAGLPRKPGMT REDLLSMNAGIVREVTG 103
E. coli IPTAVKIKGFSGEDATPALEGADVVLISAGVARKPGMD RSDLFNVNAGIVKNLVQ 103
Trypanosoma brucei CSFPLLDGVVITDEPKVAFDKADIAILCGAFPRKPGME RRDLLQTNAKIFSEQGR 115
Homo sapiens CALPLLKDVIATDKEDVAFKDLDVAILVGSMPRREGME RKDLLKANVKIFKSQGA 114
. . * :: : . * **: * :. .

P. falciparum (LDH) HIKKNC-PNAFIIVVTNPVDVMVQLLHQHS GVPKNKIIGLGGVLDTSRLKYYISQKL 167
P. falciparum (MDH) SVKLHC-SKAFVICVSNPLDIMVNVFHKFS NLPHEKICGMAGILDTSRYCSLIADKL 159

Rickettsia provazakii NIKKYA-QNAFVIVITNPLDIMVYVMLKES GLPHNKVIGMAGVLDSSRFNLFLAKEF 162
Rhizobium leguminosarum GIKKYA-PNAFVICITNPLDAMVWALQKFS GLPANKVVGMAGVLDSSRFRLFLAKEF 161
Cryptosporidium parvum NVGKYC-PNAFVICITNPLDAMVYYFKEKS GIPANKVCGMSGVLDSARFRCNLSRAL 160
Chlorobium tepidum RIMEHS-KNPIIVVVSNPLDIMTHVAWQKS GLPKERVIGMAGVLDSARFRSFIAMEL 159
E. coli QVAKTC-PKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELK 162
Trypanosoma brucei VLGEVASPNCRVCVVGNPANTNALILLRESK GKLNPRFVTALTRLDHNRATAQVAERA 173
Homo sapiens ALDKYAKKSVKVIVVGNPANTNCLTASKSAP SIPKENFS-CLTRLDHNRAKAQIALKL 171
: . . : : ** : . . . ** * ::

P. falciparum (LDH) NVCPRDVN-AHIVGAHGNKMVLLKRYITVGGIPLQEFINNKLISDAELE-AIFDRTVNTA 225
P. falciparum (MDH) KVSAEDVN-AVILGGHGDLMVPLQRYTSVNGVPLSEFVKKNMISQNEIQ-EIIQKTRNMG 217
Rickettsia prowazakii KVSVKNVN-SIVLGGHGDTMVPLLRYSTISGVPIPDLIKMGLSSNKNIE-KIIDRTKNGG 220
Rhizobium laguminosarum NVSVQDVT-AFVLGGHGDTMVPLARYSTVGGIPLTDLVTMGWVTKERLE-EIIQRTRDGG 219
Cryptosporidium parvum GVKPSDVS-AIVVGGHGDEMIPLTSSVTIGGILLSDFVEQGKITHSQIN-EIIKKTAFGG 218
Chlorobium tepidum GVSMQDVT-ACVLGGHGDAMVPVVKYTTVAGIPVADLIS AERIA-ELVERTRTGG 212
E. coli GKQPGEVE-VPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEVVEAKAGGG 221
Trypanosoma brucei RARVEEVKNCIIWGNHSGTQVPDVNSATVG GK PARAAVDNDAFFDNEFITIVQERG 229
Homo sapiens GVTANDVKNVIIWGNHSSTQYPDVNHAKVKLQGKEVGVYEALKDDSWLKGEFVTTVQQRG 231
:* * * . :

P. falciparum (LDH) LEIVNLHA—-SPYVAPAAAIIEMAESYLKDLKKVLICSTLLE-G-QYGHSD-IFGGTPVV 280
P. falciparum (MDH) AEIIKLAK-ASAAFAPAAAITKMIKSYLYNENNLFTCAVYLN-G-HYNCSN-LFVGSTAK 273
Rickettsia prowazakii GEIVKLLKTGSAYYAPAASAIAMLESYLKDKRQILTCAAYLQ-G-EYDIHD-LYIGVPII 277
Rhizobium laguminosarum AEIVGLLKTGSAYYAPAASAIEMAESYLKDKKRVLPCAAHLS-G-QYGVKD-MYVGVPTV 276
Cryptosporidium parvum GEIVELLKTGSAFYAPAASAVAMAQAYLKDSKSVLVCSTYLT-G-QYNVNN-LFVGVPVV 275
Chlorobium tepidum AEIVNHLKQGSAFYSPATSVVEMVESIVLDRKRVLTCAVSLD-G-QYGIDG-TFVGVPVK 269
E. coli S ATLSMGQAAARFGLSLVRALQGEQGVVECAYVE G DGQYARFFSQPLL 269
Trypanosoma brucei AEIMKLRGLSSALSAAKAIVDHVHDWMLGTPSGTHVSMAVYSDGNPYGVPGGLIFSFPVT 289
Homo sapiens AAVIKARKLSSAMSAAKAICDHVRDIWFGTPEGEFVSMGVISDGNSYGVPDDLLYSFPVV 291
: . : : . *. . .

P. falciparum (LDH) LGANGVEQVIELQ-LNSEEKAKFDEAIAETKRMKALA 316
P. falciprum (MDH) INNKG-AHPVEFP-LTKEEQDLYTESIASVQSNTQKAFDLIK 313
Rickettsia prowazakii IGKEGVIKVIELQ-LTEEEKILFYKSVTEVKKLIDTIQ 314
Rhizobium laguminosarum IGAGGVERIIEID-LNKTEKEAFDKSVGAVAGLCEACINIAPALK 320
Cryptosporidium parvum IGKNGIEDVVIVN-LSDDEKSLFSKSVESIQNLVQDLKSLNL 316
Chlorobium tepidum LGKNGVEHIYEIK-LDQSDLDLLQKSAKIVDENCKMLDASQG 310
E. coli LGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGEEFVNK 312
Trypanosoma brucei CSGGEWQIVSGLN-VTPAISERIKATTTELEEERREVSA 327
Homo sapiens IKNKTWKFVEGLP-INDFSREKMDLTAKELTEEKESAFEFLSSA- 334
Fig. 1. Multiple sequence alignment of MDHs from some representative organisms with deduced amino acid sequence of Pf MDH,foranalysisof
conserved motifs a nd ami no a cids impo rtant for enzyme function. MDH sequences [P. falciparum (AAQ23154.1), Rick ettsia prowazaki (NP_220759),
R. leguminosarum (CAA05717), C. parvum (AAP87358), C. tepidum (CAA56810), E. coli (NP_312136), T. brucei (AAK83037) and H. sapiens
(NP_005908)] were aligned by
CLUSTAL W
( u sing a default parameter. The sequence of Pf LDH (Q27743)
was also included in the multiple sequence align ment as it shows significant sequence similarity to Pf MDH. The N-terminal gly cine motif (the
nucleotide binding site) and the substrate binding motif have been b oxed. Other conserved amino acids are indicated by : or *.
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3493
Structural characteristics of
Pf
MDH
The model structure of Pf MDH was validated using several
tools. The Ramachandran plot [38] showed a normal
distribution of points with Phi (/) a ngles mostly r estricted to
negative values and Psi (w) values clustered in a few distinct
regions with 95% of residues occupying the allowed region.
An average value of )0.20 kT of the
MATCHMAKER
score

suggested a reasonable 3D model.
PROSTAT
check for bond
lengths, C-a chirality, amide torsion (x), Phi and Psi
torsions for helices, P hi for prolines and side chain torsions
(v
1
and v
2
) showed no major d eviation from the corres-
ponding allowed values.
PROFILES
-3
D
analysis did not
suggest any misfold and the overall self-compatibility score
for the model was 137 as compared with a score of 64 or
less; the latter would indicate an almost certainly incorrect
structure.
The model structure (Fig. 5) reveals characteristic fea-
tures of a 2-hydroxyacid dehydrogenase like MDH and
LDH including the classical Rossmann fo ld [39] constituting
the NADH binding pocket. The structural comparison of
some of the relevant 2-hydroxyacid dehydrogenases is
provided in Table 2 . The overall structure of Pf MDH is
quite distinct from that of E. coli MDH a s evidenced by
an rmsd of 5.3 between the C a coordinates which is not
surprising considering the sequence identity o f only 26%
between the two enzymes. On the other hand the structure
appears to be similar to that o f Pf LDH with a rmsd of 0.8

between the Ca atoms (Fig. 5). Both the cofactor and the
substrate binding regions are seen to overlap closely in the
two structures. However, a significant difference is observed
between the structures o f the substrate specificity loop s
(residues 78–94 of Pf MDH) as clearly seen in Fig. 5. This is
primarily due to the i nsertion of five residues into the loop in
thecaseofPf LDH (Fig. 1). The substrate specificity loop
of Pf MDH s hares a sequence identity of 65% with that of
the MDH from C. tepidum and E. coli. As expected, t he
structure of this loop in Pf MDH closely resembles that of
ligand bound activated E. coli MDH structures. The model
structure of the Pf MDH c omplexed with NADH and OAA
(Fig. 5) appears to be stabilized by several hydrogen bonds
between the e nzyme, the s ubstrate a nd the c ofactor i n
addition to hydrophobic interactions. Important residues
lining the cofactor binding pocket indicates that Gln11,
Asp32, Thr76, Ala77, Val117 and M142 are involved i n
hydrogen bonding interaction w ith NADH. Structurally
equivalent residues in the case of Pf LDH and E. coli MDH
structures show similar interactions.
The binding of OAA to the enzyme appears to be
stabilized by strong electrostatic interactions. The catalytic
His–Asp pair ( His174 and Asp147 in Pf MDH) conserved
in the 2-hydroxyacid dehydrogenase family that functions
as proton relay system appears to be oriented in a fashion
similartothatinthePf LDH (Fig. 6) and the E. coli MDH
structures. The substrate forms hydrogen bonds with
Arg81, Arg87, Asn119, His174 and Arg150 as shown in
Fig. 6A. Pf LDH appears to form a similar hydrogen
bonding network with o xamate e xcept that there is no

corresponding residue in the binding pocket equivalent to
Arg81 of Pf MDH (Fig. 6B). It is well established that the
extra conserved arginine (Arg81), present on the substrate
specificity loops of all MDHs, provides the complementary
0.1
T.brucei
Human
MOUSE
Maize
A.thaliana
T. vagi n alis
ldh-T.vagi nalis
ldh-E.coli
ldh-S.meliloti
ldh-M.loti
L.major
E.coli
ldh-Human
ldh-A.thaliana
ldh-MAIZE C.tepidum
ldh- P. falciparum
ldh-P. yoelii
P.falciparum
P. y o e l i i
R.prowazekii
M.loti
S.meliloti
R.leguminosarum
Fig. 2. Phy loge netic tree showing the evol utionary relation ship of
Pf MDH with v arious MDHs and LDHs. The unrooted tree was con-

structed by multiple alignments of all the sequences u sing
CLUSTAL X
interface with d efault paramete rs. Misaligned or poorly aligned
sequences were manually selected and realigned before constructing
the phylogenetic tree.
66
45
36
29
24
123 4567
Fig. 3. Overexpression and purification of recombinant Pf MDH.
E. c oli cultures were induced by 0.5 m
M
IPTG for 4 h at 37 °C.
Recombinant Pf MDH was purified by Ni–NTA columns (Qiagen)
according to the manufacturer’s protocol. Different protein samples
were analysed by SDS/PAGE and staining with coomassie Brilliant
blue R. Lane 1, Mole cular mass marker p roteins; lane 2, soluble ex-
tracts of recombinant E. coli lysate loaded on a Ni–NTA agarose
column; lane 3, unadsorbed proteins in the bacterial lysate passed
through a Ni–NTA agarose column; lanes 4 and 5, fractions obtained
by washing t he co lumn with the buffer containing a l ow co ncen tration
(10 m
M
) of imidazole; lane 6, rec ombinant Pf MDH protein eluted
from a Ni–NTA agarose column in buffer cont aining 200 m
M
imi-
dazole;lane7,secondeluateofthe column with buffer containing

200 m
M
inmidazole.
3494 A. K. Tripathi et al. (Eur. J. Biochem. 271) Ó FEBS 2004
charge that neutralizes the additional carboxylate group on
the substrate and therefore confers substrate specificity to
MDH.
Expression of
Pf
MDH during asexual intraerythrocytic
schizogony
Highly synchronized P. falciparum cultures were harvested
at different developmental stages and expression of
Pf MDH w as evaluated by a nalysis of transcripts using
Northern blotting (Fig. 7A) and t he enzyme protein by
Western blotting techniques (Fig. 7B). MDH in P. falcip-
rum cultures was expressed as a single transcript of
approximately 1.6 kb. The level of Pf MDH transcript
was highest in late rings and early trophozo ites which
declined further in schizonts and was very low in early
rings. Western blotting analysis, with anti-Pf MDH IgG,
detected only a single protein band of  34 kDa in
P. falciparum lysates. The level of Pf MDH protein was
equally high in the trophozoite and schizont stages of
P. falciparum while these were very low in the early and
late ring stages. The results indicate that expression of
Pf MDH is developmentally regulated. Despite significant
structural similarity, t he polyclonal a ntibodies against
Pf MDH did not cross react with Pf LDH.
Effect of the inhibitors on

Pf
MDH and
Pf
LDH
Oxamic acid, which is known to inhibit LDH by interacting
with the substrate binding site, inhibited Pf LDH as w ell as
bovine heart LDH with almost the same efficiency (Fig. 8
and Table 3). Pf MDH, porcine heart MDH (mitochon-
drial) and porcine heart M DH (cytosolic) were not inhibited
by oxamic acid up to a concentration of 2 m
M
(Table 3 and
Fig. 8). Oxamic acid (up to 1 m
M
) did not show any effect
on the growth of P. falciparum in in vitro culture. Gossypol,
which is known to inhibit Pf LDH by interacting with the
nucleotide binding site, inhibited Pf LDH with a 50%
inhibitory concentration (IC
50
)of3.1l
M
while bovine heart
LDH was much less sensitive to inhibition with gossypol.
MDHs including Pf MDH and porcine heart MDHs (both
mitochondrial and cytosolic) were inhibited by gossypol
with almost the same sensitivity (Fig. 8 and Table 3).
Gossypol inhibited the growth of P. falciparum with an
IC
50

of 11.5 ± 2.1 l
M
. Berberine, a specific inhibitor o f
cytosolic as well as organellar MDHs, did not inhibit
Pf MDH activity up to a concentration of 10 m
M
(data
not shown).
0
1000
2000
3000
4000
5678910
0
250
500
750
6 8 10 12
OAA reduction
Malate Oxidation
pH
MDH activity
Fig. 4. Ass ay of Pf MDH activity at different pH. The assay was per-
formed at different pH in phospha te (pH 5.5–8.00) and glycine
(pH 8 .00–11.00) buffers as described in the Materials and methods
section. The amount of enzyme used for assay of malate oxidation
(using NAD cofactor) was five times higher when compared to that
used for the assay of OAA reduction (using NADH cofactor). The
MDH activity is presented as lmoles of NAD red uced or (NADH

oxidized)Æmin
)1
Æ(mg protein)
)1
.
Table 1. Kinetic characterization of recombinant Pf MDH. Kinetic characteristics of the recombinant Pf MDH were determined as describe d in
Materials and methods. The specific activity is exp ressed as lmolNADHoxidizedor(NADreduced)Æmin
)1
Æmg
)1
enzyme protein. Specific activities
were calculated from the activity of enzyme at saturating concentration of different substrates and cofactors. Values are given as mean ± SD of
at least three observations .
Substrate/cofactor K
m
(m
M
)k
cat
(1Æs
)1
)k
cat
/K
m
(
M
)1
Æs
)1

) Specific activity
Reduction (pH 7.5)
Oxaloacetate (NADH) 0.030 ± 0.001 960 ± 68 32 · 10
6
3657 ± 450
NADH (OAA) 0.036 ± 0.002 950 ± 79 26 · 10
6
4870 ± 398
APADH (OAA) 0.022 ± 0.001 1010 ± 150 45 · 10
6
4935 ± 238
a-Keto-malonate (NADH) 5.44 ± 0.57 674 ± 35 0.12 · 10
6
3899 ± 390
Oxidation (pH 10.2)
Malate (NAD) 1.350 ± 0.024 250 ± 19 0.18 · 10
6
571 ± 38
NAD (malate) 0.152 ± 0.013 150 ± 20 0.98 · 10
6
507 ± 43
APAD (malate) 0.370 ± 0.019 175 ± 21 0.47 · 10
6
562 ± 56
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3495
Comparative expression of
Pf
MDH,
Pf
LDH and

Pf
MQO
and effect of gossypol
The effect of gossypol on the e xpression of Pf LDH a nd
Pf MDH in P. falciparum cultures was evaluated. Highly
synchronized cultures of P. falciparum with  15% para-
sitaemia were treated with 25 and 100 lgÆmL
)1
gossypol
and the cultures were harvested after 8 h. Control cultures
without tr eatment were s et up in parallel. Comparative
expression of Pf MDH, Pf LDH and Pf MQO were eval-
uated using the Northern blotting technique (Fig. 9A). The
control P. falciparum cultures (late trophozoite stages)
exhibited the highest expression of Pf LDH followed by
the expression of Pf MQO which was significantly lower
than Pf LDH but was still considerably higher compared to
Pf MDH. The cultures treated with 25 lgÆmL
)1
of gossypol
showed significant induction of expression of Pf MDH ,
while at this concentration expression of Pf LD H and
Pf MQO remained unaltered. Treatment with 100 lgÆmL
)1
gossypol caused further induction of Pf MDH expressio n
while expression of Pf LDH s ignificantly decreased with
this treatment (Fig. 9A). The Pf MQO expression level
remained unchanged upon treatment w ith 100 mgÆmL
)1
gossypol. Induction of Pf MDH due to gossypol treatment

was further confirmed by Western analysis (Fig. 9B).
Discussion
During asexual intraerythrocytic schizogony, th e malaria
parasite obtains its energy mainly by utiliz ation of glucose
Fig. 5. Model structure of Pf MDH superimposed on the c rystal structure o f Pf LDH. Pf L DH is shown as a white ribbon, Pf MDH as a yellow
ribbon, NADH a nd oxamate b ound to Pf LDH a s cyan sticks, N ADH and OAA bound to Pf MDH as magenta sticks. To highlight th e differences
the substrate specificity loop is coloured green in the case of Pf LDHandredinthecaseofPf MDH.
Table 2. Structural comparison of 2-hydroxyacid d ehydrogenases rela-
ted t o Pf MDH. Root mean square distanc e (rm sd) for the C a atoms is
used to compare the protiens’ structures [37].
Pf MDH
a
Pf LDH
b
C. tepidum
MDH
c
E. coli
MDH
d
T. gondii
LDH
e
Pf MDH 0 0.80 0.85 5.25 0.95
Pf LDH 0.80 0 0.93 3.30 0.79
C. tepidum
MDH
0.85 0.93 0 2.50 2.11
E. coli MDH 5.25 3.30 2.50 0 1.74
T. gondii LDH 0.95 0.79 2.11 1.74 0

a
Model structure.
b
Dunn et al. [14].
c
Dalhus et al. [23].
d
Hall and
Banaszak [35].
e
Kavanagh et al. [36].
3496 A. K. Tripathi et al. (Eur. J. Biochem. 271) Ó FEBS 2004
through g lycolysis [ 1], w hich also supplies precursors for
several other pathways i.e. nonmevalonate pathway of
isoprenoids biosynthesis, fatty acid biosynthesis, purine
salvage, pyrimidine biosynthesis, shikimate pathway and
synthesis o f GPI anchors [4]. T he parasite, however, is
equipped w ith t he complete battery of TCA cycle enzymes,
although the operation of a mitochondrial TCA cycle and
its role in energy g eneration in malaria parasites is still
debatable. A recent report has indicated the operation of
oxidative phosphorylation i n P. yoelii, a rodent malaria
parasite [5]. MDH is an important link between glycolysis,
the TCA cycle and the aspartate malate/OAA shuttle [7].
The differential MDH functions are achieved through
multiple MDH isoenzymes with different subcellular local-
izations [8]. Howeve r, the P. falciparum genome contains
only one full length MDH gene on chromosome 6 . The
Pf MDH gene did not posses any targeting signal and
therefore should be localized to the cytosol. The results

presented here, and in a recent report [40], clearly demon-
strate that this gene is functional in malaria parasites and
encodes an NAD
+
-dependent MDH. Another g ene (PF13–
0144) identified on chromosome 13 of P. falciparum as a
putative oxidoreductase, shows 49% identity with 59% of
Pf MDH and also with Pf LDH ().
This gene, however, seems to be truncated at the N-terminal
end. Pf LDH also is localized on chromosome 13 of
P. falciparum.
The b iochemical properties of r ecombinant Pf MDH are
similar to those reported earlier for the enzyme purified
from P. falciparum extracts [19]. The important character-
istic of Pf MDH is its high homology with bacterial MDHs
and a lso with L DHs. Pf MDH also s hows some distinct
enzymatic characteristics, i.e. strict substrate specificity,
insensitivity to inhibition by high concentration of the
Fig. 6. Important r esidues i nteracting with (A) OAA in Pf MDH and (B) oxamate in Pf LDH [ 9]. Th e ligands a re coloured by atom types whereas the
protein residues are shown as yellow sticks. Hydrogen bonds are d epicte d as w hite dotted lines. Hydrogen atoms are not shown f or clarity. The
residues of the Pf LDH structure are numbered in accordance with Fig. 1.
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3497
substrate and utilization of APAD as an alternate cofactor.
Some cytosolic MDHs show broader substrate specificity
and are able to utilize other dicarboxylic ke to acids also a s
the substrates [41] and MDHs are inhibited by high
concentrations of OAA [42]. Pf M DH was therefore
different from these MDHs in its biochemical characteris-
tics. Pf LDH was reported earlier to use APAD as an
alternate cofactor. This distinct characteristic of the enzyme

has been used extensively in a Pf LDH assay-based m alaria
diagnostic test, OptimalÒ [43]. The MalstatÒ reagent,
which is used for selective assay of Pf LDH in P. falciparum
cultures, is also used for quantificatio n of growth o f the
malaria parasite in an in vitro antimalarial assay [44,45].
Pf LDH utilizes APAD(H) w ith muc h higher e fficiency than
NAD(H), while Pf MDH utilizes APAD(H) and NAD(H)
with almost equal efficiency. A commercially available
NAD
+
(H) specific MDH f rom porcine heart (Sigma M
2634) did not utilize APAD(H) even up to 100 m
M
.
Pf MDH contains almost all of the amino acids residues,
which are typically conserve d in cytosolic MDHs [46]. R81,
R87 and R150, which line the anion binding site of the
substrate binding pocket, are positio ned to both stabilize
and orient t he substrate for catalysis. The D147 and H174
pair, which corresponds to the D150 and H177 pair of
E. coli MDH, may function as a proton relay system for
catalysis [22]. It h as been proposed that the presence of an
extra arginine residue (R81 in the case of Pf MDH)
provided substrate specificity to the MDH [22,23,47]. The
structure of the corresponding region in Pf LDH is highly
different, which f orms a d istant loop due to the insertion of
five amino acids [14,15]. The N-terminal glycine motif
GXGXXG is similar to the cofactor binding motif found in
most of the LDHs and a-proteobacterial MDHs [12,13,48].
In addition, G10, Q11, D32, T76, A77, V117 and M142

may be involved in a hydrogen bonding interaction with
NADH. The hydrogen bond between the carbonyl o xygen
of M142 and one of the carboxyamide hydrogens is
interesting as a similar hydrogen bond is observed with
the corresponding residue L150 (numbered as L163 in the
crystal structure) in the case of the Pf LDH crystal structure.
This hydrogen bond is considered to be a unique charac-
teristic of Pf LDH [14], as it is not seen in most other LDH
structures. Even in case of E. coli MDH structures
[22,23,35] a corresponding hydrogen bond with V146 is
not observed. Comparison of the Pf LDH crystal structure
with the Pf MDH model structure indicates that the
1.9 kb
1.39 kb
36.6 KD
a
28 KDa
ER LR ET LT ES LS
A
B
Fig. 7. Ex pressio n of Pf MDH in P. falciparum cultures during intra-
erythrocytic schizogony. (A) Northern blot analysis. Equal amounts
(20 lg) of total RNA, isolated from infected RBCs harvested at six
time points from a highly synchronized culture of P. falciparum were
electrophoresed on agarose gel, transferred t o p ositively c harged ny lon
membrane and hybridized to a radiolabelled Pf MDH probe. Mem-
branes were exposed overnight at )80 °C with radiography film to
detect the tran scripts. (B) We stern b lot analysis. T he s oluble prote in
lysates prepared from the RBC parasite preparation were separated by
SDS/PAGE (10% acrylam ide) a nd tran sfe rred t o a nitroc ellulo se

membrane. T he Pf MDH enzyme protein was detected by b lotting the
membranes with anti-Pf MDH sera (1 : 100) and detection by the
peroxidase method. ER, Early ring; LR, late ring; ET, early troph-
ozoite; L T, late trophozoite; ES, early schizont; LS, late schizont.
0
20
40
60
80
100
120
0.1 1 10 100 1000
0
20
40
60
80
100
120
1 10 100 1000 10000
Gossypol
Oxamate
Inhibitor Concentration (
µ
µµ
µ
M)
Enzyme activity remaining (%)
Fig. 8. In hibition of mammalian and Pf LDH and Pf MDH activities by
(A) gossypol and (B) oxamic acid. LDHs were assayed in the presence

of a saturating con centration of pyruvate and NADH, while MD Hs
were assayed i n th e presence o f saturating c oncentrations of OAA and
NADH. For inhibition studies the reaction mixtures containing the
enzyme and the inhibitors were preincubated f or 10 min before i niti-
ating the reaction by addition of NADH. Bovine heart LDH (j/solid
line); Pf LDH ( d/broken line); Pf MDH (h/solid line); porcine heart
MDH (mitochondrial) (s/solid line); porc ine heart MDH (cytoso lic)
(n/solid line).
Table 3. Inhibition of P. falciparum and porcine MDH and LDH by
oxamate and gossypol. The enzyme a ssays were performed with vary-
ing c oncentration s of the inhibitors, at saturating conc entrations of the
OAA (for MDH) or pyru vate (for LDH) and NADH. Values g iven
are mean ± SD of at least three observations.
Enzyme
Inhibitor IC
50
(l
M
)
Oxamate Gossypol
Pf MDH > 2000 1.5 ± 0.5
Porcine mitochondrial MDH > 2000 2.4 ± 1.1
Porcine cytosolic MDH > 2000 2.9 ± 0.9
P. falciparum LDH 58.0 ± 8.9 3.2 ± 0.7
Porcine heart LDH 78.4 ± 17.9 84.5 ± 9.5
3498 A. K. Tripathi et al. (Eur. J. Biochem. 271) Ó FEBS 2004
nucleotide binding pockets of Pf LDH and Pf MDH are
similar and explain the use of APAD(H) as an alternate
cofactor by both of the enzymes.
Expression of Pf MDH seems to be developmentally

regulatedincontrasttoPf LDH which is consistently
expressed at very high levels throughout the asexual
intraerythrocytic development of t he malaria parasite [49].
Transcription of Pf MDH is initiated during the ring
stage, peaking at the early trophozoite stage and decreas-
ing in late schizonts, while the Pf M DH protein level was
equally high in trophozoites and schizonts but was
markedly lower in rings. Both anabolic as well as
catabolic activities peak during the trophozoite stage,
subside in late schizonts and are minimal the in ring stage
[49,50]. Expression of Pf MDH therefore correlates with
the metabolic profile of the parasite. U nder normal
physiological conditions the expression of Pf MDH is
markedly lower than that of Pf LDH. Expression of
Pf MQO, another enzyme involved in t he oxidation of
malate and suggested to be localized to mitochondria, was
also significantly higher as compared t o Pf MDH .
Recently biochemical e vidence has been presented d em-
onstrating the presence of a rotenone insensitive ma-
late : quinone oxidoreductase in P. yoelii [5].
The optimum pH for reduction of OAA by Pf MDH was
found to be 7.0 while the optimum pH for malate oxidation
was highly alkaline (pH 10.2). The saturating concentration
for OAA was only 250 l
M
while for malate it was quite high
(20 m
M
). Malate oxidation at physiological pH is of
interest, but at physiological pH the reaction reaches steady

state very soo n producing only undetectable changes. At pH
7 and 7.5 no oxidation of malate could be detected, even
with very h igh concentration of the enzyme protein a nd the
substrates. As protons are the product of this reaction, the
free energy difference is highly positive a t physiological pH.
To have the reaction p roceed in the d irection of oxidation of
malate, the ratio o f OAA to malate should be very small.
Malate oxidation therefore could be observed at pH > 9.5
and only with high concentrations of malate [51]. A gene
encoding a unique MDH known as malate : quinone
oxidoreductase (MQO) was also identified on ch romosome
6ofP. falciparum (). MQO cata-
lyses the conversion of malate to OAA without NAD
+
and
uses quinone as the electron acceptor instead of NAD(P).
This dramatically reduces the free energy of the reaction.
MQO was originally identified in Crynebacterium gluta-
micum (and subsequently reported in several other bacteria
including Mycobacterium [52,53]. However in the malaria
parasite Pf MQO is functional only in combination w ith
Pf MDH, which is the sole source of malate. Pf MQO may
also be part of the suggested TCA cycle in the malaria
parasite. Degradation o f large amounts of haemoglobin by
malaria parasite during intraerythrocytic proliferation
makes it h ighly vulnerable to oxidative dam age [1]. To
avoid this oxidative damage, the parasite maintains a low
level of oxygen tension. Under these conditions oxidation of
malate by NAD
+

dependent M DH will be highly unfa-
vourable. MQO can provide an alternative route for
oxidation of malate by the malaria parasite.
Pf LDH has been exploited as a potential target for new
antimalarial drug discovery. Pf LDH activity is inhibited by
oxamic acid [14,15] ) a substrate analogue – and gossypol
[18,24] which interacts with cofactor binding site of the
enzyme. The lack of inhibition of Pf MDH by oxamate
Control
G25
G100
0
50
100
150
200
250
300
1
MDH
LDH
A
Control G25 G100
B
Control G25 G100
MDH
MQO
0
50
100

150
200
250
MDH LDH MQO
Control
Control
Control
G25
G25
G25
G100
G100
G100
Expression (% of c ontrol)Expression (% of control)
Fig. 9. Effect of treatment of P. falciparum cultures with gossypol on comparative e xpression of Pf MDH, Pf LDH and Pf MQO. Highly syn-
chronized cultures with approximately 15% parasitaemia were exposed to 25 and 100 lgÆmL
)1
gossypol at the early trophozoites stage. The
cultures were harvested after 8 h of exposure at the late trophozoites stage. Exp ressio n the enzymes was check ed by (A) a nalysis of equal amounts of
RNAbyNorthernblottingandalsoby(B)analysisofequalamounts of proteins by Western blotting. G25 and G100 indicate the P. falciparum
cultures treated with 2 5 and 100 lgÆmL
)1
gossypol, respectively. P. falciparum cultures without treatment (control) were also processed und er
similar conditions. The bars represent relative density of RNA/protein band as compared to controls based on quantitative image analysis.
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3499
reconfirms that the substrate binding pockets of Pf LDH
and Pf MDH are significantly different. Inhibition of
Pf LDH with gossypol was selective as bovine heart LDH
was inhibited w ith much lower potency. However Pf MDH
did not show much selectivity for inhibition by gossypol.

Both malarial and mammalian MDHs were in hibited by
gossypol with similar potency. Earlier reports also showed
inhibition of oxidoreductases, including MDHs from
human tissues [54] and from Trypanosoma cruzi [55], by
gossypol with alm ost the s ame poten cy. G ossypol also
inhibits growth of P. falciparum in v itro. Treatment of a
highly synchronized P. falciparum cultures (at the early
trophozoite stage) with gossypol did not cause any induc-
tion in Pf LDH expression; rather, expression of Pf LDH in
treated cultures was considerably reduced at higher doses of
gossypol. Fig. 10 shows a correlation between LDH and
MDH and MQO expression in the malaria parasite under
normal physiological conditions and also under conditions
of suppression of the expression of LDH. Almost 85% of
the glucose utilized by malaria parasites is converted to
lactate [1]. Therefore, constitutive expression of Pf LDH
may be already at a maximum level. Gossypol treatment
suppressed the expression of Pf LDH, rather than causing
any induction. A concomitant induction in expression of
Pf MDH in cultures treated with gossypol indicates that this
enzyme is regulated in t he parasite according t o its
physiological and metabolic requir ements; it would be of
interest to determine the mechanism o f this regulation.
Under normal physiological conditions, expression of
Pf MDH is significantly lower than that of Pf LDH .
Pf MDH therefore may not have a primary role in energy
generation through glucose utilization b y the malaria
parasite under normal physiological circumstances. How-
ever, i nduction of expression of Pf MDH caused by
treatment with a Pf LDH inhibitor indicates that besides

its r ole in production of malate/OAA for further metabolic
reactions, Pf MDH may also complement the Pf LDH
function of NAD/NADH coupling reactions and regener-
ation o f NAD
+
[14,15,40]. I n t he absence o f complete
oxidation of glucose by the malaria parasite regeneration of
NAD
+
is necessary for uninterrupted utilization of glucose
and energy g eneration through g lycolysis. Severe malaria
infection is usually associated with hypoglycemia and lactic
acidosis [3]. Despite the necessity of glycolysis for survival of
the malaria parasite and the distinct molecular character-
istics of the parasite e nzyme, the inhibitors of Pf LDH have
not yielded the expected results as potential antimalarial
agents. It would be useful therefore to develop dual
inhibitors of Pf LD H and Pf MDH for complete blockade
of energy generation through g lucose utilization i n the
malaria parasite. A comparative analysis of substrate and
cofactor binding pockets of Pf MDH and Pf LDH would be
important in the design of common inhibitors. Our results
thus indicate distinct biochemical and structural character-
istics of Pf MDH a nd also its possible importance in the
energy metabolism of the malaria parasite. Detailed inves-
tigations will be required to specifically pinpoint it role.
Acknowledgements
This work was supported b y CDC Cooperative agreements U50/
CCU418839 and UR3/CCU418652. Partial support was also obtained
from United States Department of Agriculture (USDA)-ARS, under

scientific cooperative agreement no. 58-6408-20009. We are thank ful to
Dr Rafael Balana Fouce for his critical reading of the manuscript and
useful suggestions. The information on the sequence of putative MDH
and MQO was obtained from PlasmoDB (),
which is supported by the Burroughs Wellcom e Fund.
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