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

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

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,

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

(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

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.

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 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

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),

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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.
References
1. Sherman, I.W. (1998) Malaria: Par asite Biology, Pathog enesis and
Protection (Sherman, I.W., ed.), pp. 135–143. ASM Press,
Washington DC.
2. Roth, E. Jr (1990) Plasmodium falciparum carboh ydrate metabo-
lism: a connection between host cell and parasite. Blood Cells 16,
453–460.
3. Holloway, P.A., Krishna, S. & White, N.J. (1991) Plasmodium
berghei: lactic acidosis and hypoglycaemia in a rodent model of
severe malaria; effects of glucose, quinine, and dichloroacetate.
Exp. Parasitol. 72, 123–133.
4. Gardner, M.J., Hall, N., Fung, E., W hite, O., Berriman, M.,
Hyman, R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman,
S.,Paulsen,I.T.,James,K.,Eisen,J.A.,Rutherford,K.,Selzberg,
S.L.,Craig,A.,Kyes,S.,Chan,M.S., Nene, V ., Shallom, S.J., S uh,
B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J.,
Haft, D., Mather, M.W., Vaidya, A.B., Martin, D.M.A., Fair-
lamb, F., Fraunholz, M.J., R oos, D.J., Ralph, S.A., McFadde n,
G.I., Cummings, L.M., Subramaniyan, M., Mungall, C., Venten,
J.C.,Carucci,D.J.,Hoffman,S.L., Newbold, C., Davis, R.W.,
Frasen, C .M. & Barrell, B. (2002) Genome sequenc e of the human
malaria parasite Plasmodium falciparum. Nature 419, 498–511.
5.Uyemura,S.A.,Luo,S.,Vieira,M.,Moreno,S.N.J.&Doc-
amopo, R. (2004) Oxidative phosphorylatio n a nd ro ten one-
insensitive malate- and NADH-quinone oxidoreduc tases in

Malate dehydrogenases – structure and function. Gen. Physiol.
Biophys. 21, 257–265.
7. Goward, C.R. & Nicholls, D.J. (1994) Malate dehydrogenase: a
model for structure, evolution, an d catalysis. Pro tein Sci. 3, 1883–
1888.
8. Musrati,R.A.,Kollarova,M.&Mernik,N.&Mikulasova,D.
(1998) Malate dehydrogenase: distribution, function and prop er-
ties. Gen. Physiol. Biophys. 17, 193–210.
9. Madern, D. (2002) Molecular evolution within 1-malate and
1-lactate dehydrogenase super-family. J. Mol Evol. 54, 825–840.
10. Zhu, G. & Keithly, J.S. (2002) Alpha-proteobacterial relationship
of apicomplexan lactate and malate dehydrogenases. J. Eukaryot.
Microbiol. 49, 255–261.
11. Madern, D., Cai, X., Abrahamsen, M.S. & Zhu, G. (2004) Evo-
lution of Cryptosporidium parvum la ctat e dehydrogenase from
malate dehydrogenase by a very recent event of gene duplication.
Mol. Biol. Evol. 21, 489–497.
12. Irmia, A., Vellieux, F.M.D., Madern, D., Zaccai, G., Karshikoff,
A., Tibbelin, G., Ladenstein, R., L ien, T. & Birkeland, N.K.
(2004) The 2.9A resolution crystal structure of malate d ehydro-
genase from Arch aeoglobus f ugidus: mechanism of oligomerization
and thermal stabilizatio n. J. Mol. Biol. 335, 343–356.
13. Irimia, A., Ebel, C., Madern, D., Richard, S.B., Cosenza, L.W.,
Zaccai, G. & Velliex, M.D. (2003) The oligomeric status of
Haloarcula marismortui malate dehyd rogenase are modulated by
solvent components as s hown by crustallographic a nd biochemical
studies. J. Mol. Biol. 326 , 859–873.
14. Dunn, C.R., B anfield, M.J., Bark er, J.J., Hingham, C.W., More-
ton,K.M.,Turgut-Balik,D.,Brady,R.L.&Holbrook,J.J.(1996)
The structure of lactate dehydrogenase from Plasmodium falci-

Biochem. 6, 533–535.
22. Bell, J.K., Yennawar, H.P., Wright, S.K., Thompson, J.R., Viola,
R.E. & Banaszak, L.J. (2001) Structural analyses of a malate
dehydrogenase with a variable active site. J. Biol. Chem. 276,
31156–31162.
23. Dalhus, B., Saarinen, M., Sauer, U.H., Eklund, P., Johansson, K.,
Karlsson, A., Ramaswamy, S., Bjork, A., Synstad, B., Naterstad,
K.,Sirevag,R.&Eklund,H.(2002)Structuralbasisforthermo-
philic protein stability: structures of thermophilic and mesophilic
malate dehydrogenases. J. Mol. Biol. 318, 707–721.
24.Royer,R.E.,Deck,L.M.,Campos,N.M.,Hunsaker,L.A.&
Vander Jagt, D .L. (1986) Biologically active derivatives of gossy-
pol: syn thesis and antimalarial activities of peri-acylated gossylic
nitriles. J. Med. Chem. 29, 1799–1801.
25. Trager, W. & Jenson, J.B. (1976) Human malaria parasites in
continuous culture. Science 193, 673–675.
26. Lambros, C. & Vanderberg, J.P. (1979) Synchronization of Plas-
modium falciparum erythrocytic stages in culture. J. Parasitol. 65,
418–420.
27. Elandalloussi, L.M. & Smith, P.J. (2002) Preparation of pure and
intact Plasmodium falciparum plasma membrane vesicles and
partial characterization of the plasma membrane ATPase. Malar.
J. 1, 1–7.
28. Bradford, M.M. ( 1976) A rapid and sensitive me thod for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
29. Muhammad, I., Dunbar, D.C., Khan, S.I., Tekwani, B.L., Bedir,
E., Takamatsu, S., Ferreira, D. & Walkerm, L.A. (2003) Anti-
parasitic alkaloids from Psychotria klugii. J. Nat. Prod. 66, 962–
967.

40. Chan, M. & Sim, T.S. (2004) Functional characterization of an
alternative [lactate dehydrogenase-like] malate dehydrogenase in
Plasmodium falciparum. Parasitol. Res. 92, 43–47.
41. Friedrich, C.A., Ferrell, R.E., Siciliano, M.J. & Kitto, G.B. (1988)
Biochemical and genetic i dentity of alpha-k eto acid r eductase and
cytoplasmic malate d ehydrogenase from human e rythrocytes.
Ann. Human Genet. 52, 25–37.
42. Vessal, M. & Tabei, S .M. (1996) Partial purification and k inetic
properties of cyto plasmic malate dehydrogenase from ovine liver
Echinococcus granulosus protoscolices. Com p Biochem. Phys iol. B
Biochem. Mol Biol. 113, 757–763.
Ó FEBS 2004 Malate dehydrogenase of P. falciparum (Eur. J. Biochem. 271) 3501
43. Moody, A.H. & Chiodini, P.L. (2002) N on-microscopic method
for malaria diagnosis using OptiMAL IT, a second-generation
dipstick for malaria pLDH antigen detectio n. Br.J.Biomed.Sc.
59, 228–231.
44. Piper, R., Lebras, J., Wentworth, L., Hunt-Cooke, A., Houze, S.,
Chiodini, P . & Makler, M. (1999) Immunocapture diagnostic
assays for malaria using Plasmodium lactate dehydrogenase
(pLDH). Am. J. Trop. M ed. Hyg. 60, 109–118.
45. Makler, M.T ., Ries , J.M ., Williams, J.A., Bancroft, J.E., Pipe,
R.R.C., Gibbins, B.L. & Hinrichs, D.J. (1993) Parasite lactate
dehydrogenase as an assay for Plasmodium falciparum drug sen-
sitivity. Am. J. Trop. Med. Hyg. 48, 739–741.
46. Chapman, A.D., Cortes, A., Dafforn, T.R., Clarke, A.R. &
Brady, R.L. (199 9) Stru ctural basis o f substrate specificity in
malate dehydrogenases: crystal structure of a ternary complex of
porcine cytoplasmic malate dehydrogenase, alpha-ketomalonate
and tetrahydoNAD. J. Mol. B iol. 285, 703–712.
47. Wright, S .K. & Viola, R.E. (2001) Alteration of the specificity o f

Rovai, L.E. & Blan co, A. (1984) Inhbition by gossypol of
oxidoreductases from Trypanosoma cruzi. Biochem. Pharmacol.
33, 955–959.
3502 A. K. Tripathi et al. (Eur. J. Biochem. 271) Ó FEBS 2004