Eur J Biochem 271, 3488–3502 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04281.x An a-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum Cloning and biochemical characterization of the enzyme Abhai K Tripathi1, Prashant V Desai2, Anupam Pradhan1, Shabana I Khan1, Mitchell A Avery1,2, Larry A Walker1,3 and Babu L Tekwani1 National Center for Natural Product Research, Research Institute of Pharmacological Sciences, 2Department of Medicinal Chemistry, and 3Department of Pharmacology, School of Pharmacy, University of Mississippi, MS, USA Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites The cDNA corresponding to the MDH gene, identified on chromosome of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD+(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD The enzyme exhibited strict substrate and cofactor specificity The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in a-proteobacterial MDHs Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also Treatment of a synchronized culture of P falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs The enzymes associated with carbohydrate and energy metabolism in malarial parasites have attracted significant attention as potential targets for new antimalarial drug discovery [1] During asexual reproduction and growth within the host’s erythrocytes the parasite depends mainly on the glycolytic pathway to obtain energy Infected erythrocytes consume almost 100 times more glucose than uninfected erythrocytes [2] Almost all of this increase in glucose utilization is the result of the synthesis of enzymes of glycolytic (and associated) pathways by the parasite Fulminating malaria infections are characterized by hypoglycemia and potentially lethal lactic acidosis [3] Earlier studies and the recent release of a fully annotated map of the Plasmodium falciparum genome have shown the presence of a complete battery of enzymes of the Embden–Meyerhof– Parnas pathway of glycolysis and the tricarboxylic acid (TCA) cycle in malarial parasites [4] However, the role of oxidative metabolism in the malaria parasite through the TCA cycle remains unclear A recent report has indicated the operation of oxidative phosphorylation and the presence of an alternative NADH-Q oxidoreductase and malate:quinone oxidoreductase in Plasmodium yoelii [5] The oxidative metabolism in malaria parasite may be important for de novo pyrimidine biosynthesis rather than energy metabolism [1] L-Malate dehydrogenase (MDH; EC 1.1.1.37) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) are 2-ketoacid:NAD(P) oxidoreductases that are universally distributed in both eukaryotic and prokaryotic organisms [6,7] LDH is particularly important in anaerobic metabolism, Correspondence to B L Tekwani, National Center for Natural Product Research, School of Pharmacy, University of Mississippi, MS 38677, USA Fax: +1 662 915 7062, Tel.: +1 662 915 7882, E-mail: btekwani@olemiss.edu Abbreviations: IPTG, isopropyl thio-b-D-galactoside; Pf MDH, Plasmodium falciparum malate dehydrogenase; Pf LDH, P falciparum lactate dehydrogenase; Pf MQO, P falciparum malate : quinone oxidoreductase; OAA, oxaloacetate; APAD, acetyl pyridine adenine dinucleotide; RBC, red blood cells; TCA, tricarboxylic acid Enzymes: L-Malate dehydrogenase (MDH; EC 1.1.1.37); L-lactate dehydrogenase (LDH; EC 1.1.1.27) Notes: Part of the work reported in this paper was presented at the 51st Annual Meeting of American Society of Tropical Medicine & Hygiene, held at Denver, November 10–14, 2002 [Am J Trop Med Hyg (2002) 67, 146] The cDNA sequence reported in paper has been submitted to GenBank under accession no AY324107 (Received March 2004, revised June 2004, accepted July 2004) Keywords: gossypol; lactate dehydrogenase; malate dehydrogenase; malate:quinone oxidoreductase; Plasmodium falciparum Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3489 while MDH may have a role in oxidative metabolism as well as other physiological functions, depending on its biochemical characteristics and intracellular localization [8] LDHs are cytosolic proteins but isoforms of MDH have been localized to the cytosol as well as different subcellular organelles such as mitochondria, chloroplast, peroxysomes and glyoxysomes [8] LDHs and MDHs characterized from different organisms form a large superfamily [9] A specific phylogenetic distribution of LDH, LDH-like MDH and dimeric MDH over the Archaeal, Bacterial and Eukaryal domains was observed All LDHs and MDHs enzymes from apicomplexan parasites, which include Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum and Eimeria tenella, were found to be monophylectic within the ÔLDH-like MDHÕ group as a sister to alpha-proteobacterial MDHs [10] All of the apicomplexan LDHs, with the exception of LDH1 from Cryptosporidium parvum, form a separate clade from their MDH counterparts, indicating that these LDHs evolved from an ancestral apicomplexan MDH by gene duplication and functional conversion before expansion of apicomplexans [10,11] Both LDH and MDH from various organisms have been characterized in considerable detail at the molecular and structural levels [6,7,12,13] However, only LDH from P falciparum (Pf LDH) has been characterized in significant detail, including determination of its crystal structure The enzyme has also been exploited as a potential target for design and development of specific enzyme inhibitors as antimalarial agents [14–17] A comparative kinetic and structural analysis of LDH from all four species of human malaria parasite has also been reported recently [18] However, MDH, which catalyses reversible conversion of malate to oxaloacetate (OAA) using NAD(P) as a cofactor, has not been characterized in sufficient detail from P falciparum It was shown previously that P falciparum contained a single isozyme of MDH (Pf MDH) which was suggested to be localized to the cytosol of the parasite [19] The avian malaria parasite P lophurae was also found to contain a single isozyme of MDH which was distinct from host erythrocyte MDH [20] Alternatively, P berghei the rodent malaria parasite has been shown to have multiple MDH isozymes [21] However, the presence of multiple MDH isoforms in mouse erythrocytes, the preference of P berghei to infect reticulocytes, and the detection of low activity of MDH in P berghei raises questions regarding the validity of this report The recent release of the P falciparum genome sequence has indicated the presence of a gene putatively encoding for MDH [4] Initial analysis of the Pf MDH sequence revealed its higher homology with the MDH sequences from a-proteobacteria and also with LDHs Pf MDH may therefore be classified as LDH-like MDH, similar to that from other apicomplexan parasites [10,11] In this study Pf MDH was cloned from P falciparum genomic DNA and total RNA by PCR and RT-PCR, overexpressed in Escherichia coli and the recombinant enzyme was purified to homogeneity Functional characterization of the recombinant Pf MDH protein was carried out by analysis of its biochemical characteristics and enzyme kinetics Expression of MDH in P falciparum was found to be developmentally regulated during its growth and proliferation in human erythrocytes A highly refined homology model for Pf MDH was also constructed on the basis of crystal structures of nearest structural homologues i.e Pf LDH [14], E coli MDH [22] and Chlorobium tepidum MDH [23] High homology of Pf MDH with Pf LDH also prompted us to ask whether MDH can complement the function of LDH in the malaria parasite This was studied by treating the P falciparum cultures with gossypol, an inhibitor of LDH, which has also shown antimalarial action [24] Materials and methods P falciparum (D6 strain) was grown in vitro in RPMI 1640 medium with A+ human red blood cells (RBCs) and A+ human serum as described previously [25] For large-scale culture the parasite was grown in 75 cm2 culture flasks, which can accommodate up to 200 mL of culture medium The parasite was grown in 24 culture flasks to  10–15% parasitemia The cultures were highly synchronized with two/three cycles of sorbitol treatment [26] The parasite cultures initiated with early ring stage were harvested at regular h intervals starting from to 40 h to isolate the parasite at different developmental stages of life cycle viz., early rings, late rings, early trophozoites, late trophozoites, early schizonts and late/mature schizonts The RBC-free parasite was prepared by lysis of infected RBCs with saponin [27] Genomic DNA and RNA were prepared by using a genomic DNA isolation kit (Qiagen) and trizolÒ method (Invitrogen), respectively, as per the manufacturer’s protocol Cloning of Pf MDH, Pf LDH and Pf MQO Pf MDH was cloned for functional and biochemical characterization The other two genes Pf LDH and Pf MQO were also cloned to obtain the DNA probes for analysis of its expression in P falciparum cultures Pf LDH was also overexpressed in E coli cultures to obtain the enzyme for evaluation of the inhibitors The primers (forward primer 5¢-ACTAAAATTGCTTTAATAGG TAG-3¢ and reverse primer 5¢-TTATTTAATGTC GAAAGC-3¢) for cloning of full-length MDH ORF DNA and cDNA corresponding to the complete coding sequence were designed from the putative MDH gene (MAL6P1.242) identified on chromosome of P falciparum (http://www.plasmodb.org) The DNA corresponding to full-length Pf MDH ORF was amplified by PCR using Taq DNA polymerase and P falciparum genomic DNA as the template The cDNA was prepared by RT-PCR using DNase-treated total RNA, isolated from mixed culture of P falciparum as the template using a Qiagen RT-PCR kit The control reaction without reverse transcriptase was run simultaneously to check for genomic DNA contamination and to ensure specific amplification of cDNA The DNA corresponding to full-length ORF sequence of Pf LDH (PF13-0141) and Pf MQO (MAL6P1.258) were also amplified using the appropriate primers (Pf LDH: forward 5¢-ATGGCACCAAAAGCAAAAATCG-3¢ and reverse 5¢-AGCTAATGCCTTCATTCTCTTAG-3¢; Pf MQO forward 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 3490 A K Tripathi et al (Eur J Biochem 271) (Qiagen) The ligation mixtures were used to transform E coli XL-1 blue cells and the bacterial colonies transformed with recombinant plasmids were selected on Luria–Bertani medium agar plates containing 100 lgỈmL)1 ampicillin The presence of the DNA/cDNA inserts, and their orientation in the recombinant plasmids, was confirmed by digestion of the plasmid minipreps with appropriate restriction enzymes and their analysis by electrophoresis on 0.8% (w/v) agarose gels The plasmid containing Pf MDH ORF (DNA and cDNA) and Pf MQO cDNA in the correct orientation were sent to Laragen Inc (http://www.laragen.com) for sequencing on both strands The Pf MDH mRNA sequence for P falciparum (Sierra Leone D6 strain) has been submitted to GenBank under accession no AAQ23154 and was found to be the same as that reported for a putative MDH sequence of a 3D7 P falciparum strain The recombinant pQE30Pf LDH and pQE30Pf MQO plasmids were also analysed in the same way Overexpression and purification of recombinant proteins For overexpression of Pf MDH, Pf LDH and Pf MQO the recombinant plasmids prepared from XL-1 blue cells were transformed into E coli M15 (pREP 4) cells as recommended by Qiagen The transformed colonies were selected on LB agar plates containing 100 lgỈmL)1 ampicillin and 25 lgỈmL)1 kanamycin A 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 °C to D600  0.5 At this time, isopropyl thio-b-D-galactoside (IPTG) was added to the cultures to a final concentration of 0.5 mM in order to induce overexpression of recombinant proteins The cultures were grown for additional h at 37 °C with constant shaking Cells were harvested by centrifugation at 5000 g for 15 at °C Harvested bacterial pellets were re-suspended in phosphate buffer containing 300 mM NaCl and 10 mM imidazole and lysed by sonication The extracts were centrifuged at °C at 15 000 g for 30 Expression of recombinant protein was checked by SDS/PAGE and also by Western blotting Significant overexpression of Pf MDH and Pf LDH was achieved in E coli cultures induced with IPTG Expression of recombinant Pf MDH and Pf LDH could be achieved in soluble fractions However, overexpression of Pf MQO could not be achieved in this system, even with the use of various concentrations of IPTG and varying culture conditions The soluble fractions, which contained significant amounts of the Pf MDH/Pf LDH protein, were used for purification The recombinant proteins contain a · His tag which facilitates their purification by affinity chromatography using Ni–NTA chelating columns The soluble bacterial extracts were passed through Ni–NTA agarose columns and the columns were washed with buffer containing 20 mM imidazole Recombinant proteins were eluted with buffer containing 200 mM imidazole The soluble extracts, column fractions and purified proteins were analysed by SDS/PAGE Gels were stained with Coomassie brilliant blue R and also with silver stain, to check the purity of recombinant proteins The oligomeric structure of the enzymatically functional recombinant Pf MDH preparations was determined by a standard size exclusion chromatography procedure using Sephacryl S200 Enzyme assays MDH catalyses reversible oxidation of malate to OAA utilizing NAD(H) or NADP(H) as cofactor MDH was assayed spectrophotometrically for both oxidation of malate and reduction of OAA by recording the change in absorbance at 340 nm as described earlier [19] The assays were set up in clear flat-bottomed 96-well microplates For the malate reduction assay, the reaction mixture contained in a total volume of 200 lL with glycine buffer (pH 10.2, 50 mM) malate (20 mM or as specified) and NAD or NADP (500 lM or as specified) and an appropriate amount of the enzyme Similarly for the OAA reduction assay the reaction mixture contained in a total volume of 200 lL with phosphate buffer (pH 7.0, 50 mM), OAA (250 lM or as specified) and NADH or NADPH (200 lM 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 and the change in absorbance per minute was recorded Activity of the enzyme was calculated in terms of micromoles of NAD reduced or NADH oxidized Similarly Pf LDH activity was also measured according to the spectrophotometric method as described earlier [16] For determination of optimum pH for pfMDH activity the assays were performed at different pH ranging from 6.0 to 11.0 using phosphate (pH 6.0–8.0) and glycine (pH 8.5–11.0) buffers with saturating concentrations of the substrate and the cofactor For enzyme kinetic studies, assays were performed at varying concentrations of substrates (OAA or malate) and the cofactors [NAD/ H or acetyl pyridine adenine dinucleotide (APAD/H)] Kinetic constants were computed with GRAFIT To determine substrate specificity of Pf MDH the enzyme activity was also determined using different 2-keto or 2-hydroxy carboxylic acids such as lactate, pyruvate, a-ketoglutarate, keto-malonate, oxo-butyrate and ketoadipic acid Analysis of expression of the enzymes in P falciparum cultures Expression of Pf MDH at the different stages of the asexual intra-erythrocytic cycle was evaluated by semiquantitative analysis of transcripts by Northern blotting and also by quantification of the enzyme protein by Western blotting Expression of Pf MDH, Pf LDH and Pf MQO in control and drug treated P falciparum cultures was evaluated by Northern and Western blotting Quantitative analysis of the transcripts and proteins was performed by using the NIH IMAGE (version 1.61) analysis program Northern blotting The P falciparum cultures at different stages of life cycle or the control and drug treated cultures were harvested by centrifugation Total RNA was isolated directly from the infected RBCs by the TrizolÒ method as per the manufacturer’s protocol Purity and concentration of RNA was checked spectrophotometrically by reading the Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3491 absorbance at 260/280 nm An equal amount of total RNA (20 lg) from each of the six stages or untreated and drug treated samples was separated on denaturing 1% (w/v) agarose gel To check the concentration and purity of RNA the gels were stained with ethidium bromide and visualized on a UV transluminator Equal intensity of ribosomal RNA bands in all of the lanes indicated that an equal amount of total RNA had been loaded The RNA from agarose gels was transferred under vacuum to positively charged nylon membrane (Sigma) and the membranes were washed with 0.1% NaCl/Cit and 0.1% SDS for 30 and prehybridized in 10 mL prehybridization solution [3.6 mL sonicated salmon sperm DNA of a 10 mgỈmL)1 stock, 0.2 mL 50· Denhardt’s solution, 0.1 mL 10% (w/v) SDS, 3.64 mL 10 mgỈmL)1 and mL 20· NaCl/Cit] for h at 42 °C with gentle shaking Membranes were then hybridized with hybridization solution containing denatured radioactive Pf MDH or Pf LDH or Pf MQO probe The recombinant plasmids containing Pf MDH/Pf LDH/Pf MQO cDNA inserts were digested with appropriate restriction enzymes and the inserts were purified on the agarose gel, using a gel extraction kit (Qiagen) The probes were prepared by labelling the inserts with [32P]dCTP using a random prime labelling kit (Amersham Biosciences) Hybridization was performed overnight at 42 °C with gentle shaking The membranes were then washed successively with 10· NaCl/ Cit, 1% (w/v) SDS (20 min), 1· NaCl/Cit, 0.5% (w/v) SDS (30 min) and 0.1· NaCl/Cit, 0.2% (w/v) SDS (45 min) The membranes were exposed to hyperfilm and the transcripts were visualized by autoradiography Western blotting For quantification of enzyme protein the parasites were isolated from infected RBCs by saponin lysis [27] The parasite pellets were washed extensively with cold NaCl/Pi to remove RBC proteins and membrane contaminants Finally, the parasite pellet was resuspended in NaCl/Pi containing 0.1% (v/v) Triton X-100 and the proteins were solublized by sonication The soluble protein extracts were obtained after centrifugation of the lysates at 10 000 g for 15 in an Eppendorf microcentrifuge tube Protein concentrations in the clear supernatants were determined by the Bradford method [28] Equal amount of total protein (100 lg) from each sample was loaded and separated by SDS/PAGE Prestained molecular mass protein markers (Sigma-Aldrich) were also loaded in the first lane The proteins were transferred onto nitrocellulose membrane (Sigma) using semi-dry blot and membrane was probed with purified IgG fraction obtained from polyclonal antiserum raised against recombinant Pf MDH (Antibodies Inc.) Pf MDH protein was visualized using peroxidase conjugated secondary 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 lgỈmL)1 in the culture medium The cultures were harvested after h and total RNA was isolated from the infected RBCs and analysed for expression of Pf MDH, Pf LDH and Pf MQO by Northern blotting as described above Parasite was isolated from control and treated cultures and equal amount of protein was analysed by Western blotting as described above Antimalarial activities of oxamic acid and gossypol were determined in vitro for P falciparum cultures as described earlier [29] Analysis of Pf MDH sequence Protein sequences of various MDHs and LDHs were obtained from GenBank All of the selected sequences along with Pf MDH sequence were aligned using CLUSTAL– WINDOWS interface with default parameters [30] Misaligned or poorly aligned sequences were selected manually and realigned to obtain proper alignment of all sequences PHYLIP format tree output was selected to obtain the bootstrap neighbor-joining tree An unrooted phylogenetic tree was drawn using TREE VIEW The accession number of the sequences used for the phylogenetic analysis were: P falciparum MDH (NP_703844), P falciparum LDH (CAD52397), P yoelii MDH (EAA22943), P yoelii LDH (EAA15666), Ricketssia prowozekii MDH (NP_220759), Mesorhizobium loti MDH (NP_105210), Sinorhizobium meliloti MDH (AAG41996), Rhizobium leguminosarum MDH (CAA05717), Trypanosoma brucei MDH (AAK83037), human cytoplasmic MDH (P40925), mouse cytoplasmic MDH (P14152), maize cytoplasmic MDH (T02935), Arabidopsis thaliana MDH (AAM65532), Trichomonas 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 Graphics Octane workstation, equipped with two parallel R12000 processors, V6 graphics board and 512 MB memory Comparative protein structure modelling was performed with the HOMOLOGY module of INSIGHTII 2000 (Accelrys Inc., San Diego, CA, USA) Energy minimizations 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-3D [31] modules of INSIGHTII 2000 as well as the MATCHMAKER [32] module of SYBYL 6.9 (Tripos Associates Inc., St Louis, MO, USA) A WU-BLAST 2.0 [33] PDB search was performed on the Pf MDH sequence with default parameters of BLASTP gapped alignment Only two sequences, MDH from C tepidum (40% sequence identity) and Pf LDH (39% sequence identity) passed the identity filter of 35% and hence were used to build the 3D model A total of nine structurally conserved regions and seven structurally variable regions or loops were identified The structurally conserved regions were built from the homologues whereas the coordinates for the loops were obtained by searching the PDB for regions of proteins that meet a defined geometric criterion The protocol uses an existing 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 3492 A K Tripathi et al (Eur J Biochem 271) residues present between the regions of interest Different rotamers for the residues that line the active pocket were also studied and the most energy stable rotamers were retained The crude model so obtained was then refined by minimization using an analogous approach reported previously [34] The structure of the Pf MDH in complex with OAA and NADH was obtained as follows First, the substrate and the cofactor were placed at appropriate binding regions of Pf MDH based on their corresponding locations in the Pf LDH [14] and C tepidum MDH structures [23] The complex was then minimized using steepest descents for 1000 iterations followed by 2000 iterations of conjugate gradients This was followed by molecular dynamics simulations for 25 ps at 300 K and finally minimization to ˚ a gradient of 0.001 kcalỈmol)1ỈA)1 or less using conjugate gradients During this refinement, the side chains of the residues lining the substrate as well as the cofactor binding pockets were allowed to move freely whereas rest of the protein atoms were fixed Comparison of the Pf MDH model structure with crystal structures of Pf LDH [14], C tepidum MDH [23], E coli MDH [35] and T gondii LDH [36] was achieved by determination of the rmsd between each pair This is one of the most acceptable methods for determination of structural similarity among the proteins [37] Results Cloning and characterization of Pf MDH mRNA and gene Preparation and amplification of cDNA corresponding to the complete encoding region of Pf MDH by RT-PCR yielded a single amplicon of 942 bases, which encodes a predicted protein of 313 amino acids with calculated molecular mass of 34 040 Da The sequence of Pf MDH mRNA from P falciparum (Sierra Leone D6 strain) was found to be the same as reported for the Pf MDH gene identified on chromosome of P falciparum (3D7 strain) The results confirm that the PfMDH gene had no introns Presence of Pf MDH mRNA in P falciparum cultures also indicated that the parasite expressed the gene during the asexual reproduction cycle The sequence of the Pf MDH protein was subjected to BLAST-P analysis with nonredundant GenBank protein database Comparison of Pf MDH sequence with some representative MDH sequences are presented in Fig The sequence of Pf LDH was also included in this as Pf MDH showed significant similarity with a-proteobacterial MDHs and also with LDHs characterized in bacteria and lower eukaryotes, particularly that from apicomplexan parasites Pf MDH did not possess any target signal sequence and therefore represents a cytosolic MDH This was further confirmed by the observation that Pf MDH sequence shows higher homology with cysolic MDH than with organellar MDHs (Fig 2) The Pf MDH protein sequence also contained several conserved motifs and amino acids residues that have been found to be important in MDH and LDH functions (Fig 1) The N-terminal glycine motif which is involved in nucleotide binding function was identified as GSGQIG in Pf MDH and corresponded to the signature sequence GXGXXG, found in proteobacterial MDHs and Ó FEBS 2004 LDHs, rather than the signature sequence of GXXGXXG found in most of the MDHs More detailed analysis of substrate and nucleotide binding sites has been described in the section on structural analysis of Pf MDH by homology modelling Overexpression and biochemical characterization of Pf MDH Overexpression of Pf MDH cDNA in E coli yielded a recombinant protein of  36 kDa as analysed by SDS/ PAGE (Fig 3) The recombinant protein expressed in E coli was larger then the predicted Pf MDH protein of 34 kDa because it also contained some extra amino acids derived from the bacterial expression vector, including a His6-tag at the N-terminal end, which facilitates its purification Most of the recombinant protein was recovered in the soluble fraction and was found to be functionally active as MDH, i.e it catalysed reduction of OAA and oxidation of malate in the presence of NADH/NAD The protein could be purified to apparent homogeneity, as analysed by SDS/PAGE, after single-step affinity chromatography through a Ni–NTA agarose column About 10 mg of the functionally active recombinant Pf MDH could be recovered from L of the E coli culture The homogeneous preparations of recombinant Pf MDH were used to characterize oligomeric status, enzyme kinetic properties, and substrate/cofactor specificities Analysis of recombinant Pf MDH by size exclusion chromatography on Sepahcryl S200 did not provide conclusive information on the oligomeric state of the enzymatically active protein Both dimeric as well as terameric forms of the proteins were eluted Pf MDH may exist as a dimer of dimers MDH catalyses interconversion of malate and OAA inside the cell However, in vitro these reactions occur at a different pH Therefore, before a detailed kinetic analysis of the enzyme could be conducted, the optimum pH for oxidation of malate and reduction of OAA were determined by performing the assays at varying pH (5.5–11.0) using different reaction buffers (Fig 4) The optimum pH for oxidation of malate in the presence of NAD was 10.2 and for the reduction of OAA using NADH as the cofactor it was 7.0 Further kinetic characterization of enzyme was performed at the experimentally determined pH optima Pf MDH catalysed the reduction of OAA with an efficiency six to eight times greater than that of oxidation of malate as determined by evaluation of Vmax and kcat values for malate/NAD and OAA/NADH (Table 1) The saturating concentrations of OAA and NADH were found to be  250 lM and  150 lM, respectively, while for malate and NAD these were  20 mM and  1.5 mM, 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 mM A surprising observation was the use of acetyl pyridine adenine dinucleotide (APAD/ APADH) as an alternate cofactor by Pf MDH, which showed almost comparable efficiency to that of NAD/ NADH (Table 1) Porcine heart MDH (M7383, SigmaAldrich) did not show any activity with APAD/APADH Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3493 P falciparum(LDH) P falciparum(MDH) Rickettsia prowazekii Rhizobium leguminosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens MAPKAKIVLV-G-SGMIGGVMATLIVQKNLG -DVVLFDIVKNMPHGKALD TSHT -MTKIALI-G-SGQIGAIVGELCLLENLG -DLILYDVVPGIPQGKALD LKHF MKKNPKISLI-G-SGNIGGTLAHLISLKKLG -DIVLFDVSEGLPQGKALD LMQA MARN-KIALI-G-SGMIGGTLAHLAGLKELG -DIVLFDIADGIPQGKGLD ISQS MR KKISII-G-AGQIGSTIALLLGQKDLG -DVYMFDIIEGVPQGKALD LNHC MKITVI-G-AGNVGATTAFRLAEKQLAR ELVLLDVVEGIPQGKALD MYES MK-VAVLGAAGGIGQALALLLKTQLPSG-SELSLYDIAPVTP-GVAVD -LSH MSNTCKRVAVTGAAGQIGYSLLPLIAAGRMLGFDQRVQLQLLDISPALKALEGIRAELMD MSEPIR-VLVTGAAGQIAYSLLYSIGNGSVFGKDQPIILVLLDITPMMGVLDGVLMELQD : * :* : : : * :: 51 48 51 50 49 48 48 60 59 P falciparum (LDH) P falciprum (MDH) Rickettsia provazakii Rhizobium legumunosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens NVMAYSNCKVSGSNTYDDLAGADVVIVTAGFTKAPGKSDKEWNRDDLLPLNNKIMIEIGG STILGVNRNILGTNQIEDIKDADIIVITAGVQRKEGMT -REDLIGVNGKIMKSVAE ATIEGSDIKIKGTNDYRDIEGSDAVIITAGLPRKPGMS -RDDLISVNTKIMKDVAQ SPVEGFDVNLTGASDYSAIEGADVCIVTAGVARKPGMS -RDDLLGINLKVMEQVGA MALIGSPAKIFGENNYEYLQNSDVVIITAGVPRKPNMT -RSDLLTVNAKIVGSVAE GPVGLFDTKVTGSNDYADTANSDIVVITAGLPRKPGMT -REDLLSMNAGIVREVTG IPTAVKIKGFSGEDATPALEGADVVLISAGVARKPGMD -RSDLFNVNAGIVKNLVQ CSFPLLDGVVITDEPKVAFDKADIAILCGAFPRKPGME -RRDLLQTNAKIFSEQGR CALPLLKDVIATDKEDVAFKDLDVAILVGSMPRREGME -RKDLLKANVKIFKSQGA * :: : * **: * : 111 103 106 105 104 103 103 115 114 P falciparum (LDH) P falciparum (MDH) Rickettsia provazakii Rhizobium leguminosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens HIKKNC-PNAFIIVVTNPVDVMVQLLHQHS -GVPKNKIIGLGGVLDTSRLKYYISQKL SVKLHC-SKAFVICVSNPLDIMVNVFHKFS -NLPHEKICGMAGILDTSRYCSLIADKL NIKKYA-QNAFVIVITNPLDIMVYVMLKES -GLPHNKVIGMAGVLDSSRFNLFLAKEF GIKKYA-PNAFVICITNPLDAMVWALQKFS -GLPANKVVGMAGVLDSSRFRLFLAKEF NVGKYC-PNAFVICITNPLDAMVYYFKEKS -GIPANKVCGMSGVLDSARFRCNLSRAL RIMEHS-KNPIIVVVSNPLDIMTHVAWQKS -GLPKERVIGMAGVLDSARFRSFIAMEL QVAKTC-PKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELK VLGEVASPNCRVCVVGNPANTNALILLRESK GKLNPRFVTALTRLDHNRATAQVAERA ALDKYAKKSVKVIVVGNPANTNCLTASKSAP SIPKENFS-CLTRLDHNRAKAQIALKL : : : ** : ** * :: 167 159 162 161 160 159 162 173 171 P falciparum (LDH) P falciparum (MDH) Rickettsia prowazakii Rhizobium laguminosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens NVCPRDVN-AHIVGAHGNKMVLLKRYITVGGIPLQEFINNKLISDAELE-AIFDRTVNTA 225 KVSAEDVN-AVILGGHGDLMVPLQRYTSVNGVPLSEFVKKNMISQNEIQ-EIIQKTRNMG 217 KVSVKNVN-SIVLGGHGDTMVPLLRYSTISGVPIPDLIKMGLSSNKNIE-KIIDRTKNGG 220 NVSVQDVT-AFVLGGHGDTMVPLARYSTVGGIPLTDLVTMGWVTKERLE-EIIQRTRDGG 219 GVKPSDVS-AIVVGGHGDEMIPLTSSVTIGGILLSDFVEQGKITHSQIN-EIIKKTAFGG 218 GVSMQDVT-ACVLGGHGDAMVPVVKYTTVAGIPVADLIS -AERIA-ELVERTRTGG 212 GKQPGEVE-VPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEVVEAKAGGG 221 RARVEEVKNCIIWGNHSGTQVPDVNSATVG GK PARAAVDNDAFFDNEFITIVQERG 229 GVTANDVKNVIIWGNHSSTQYPDVNHAKVKLQGKEVGVYEALKDDSWLKGEFVTTVQQRG 231 :* * * : P falciparum (LDH) P falciparum (MDH) Rickettsia prowazakii Rhizobium laguminosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens LEIVNLHA—-SPYVAPAAAIIEMAESYLKDLKKVLICSTLLE-G-QYGHSD-IFGGTPVV AEIIKLAK-ASAAFAPAAAITKMIKSYLYNENNLFTCAVYLN-G-HYNCSN-LFVGSTAK GEIVKLLKTGSAYYAPAASAIAMLESYLKDKRQILTCAAYLQ-G-EYDIHD-LYIGVPII AEIVGLLKTGSAYYAPAASAIEMAESYLKDKKRVLPCAAHLS-G-QYGVKD-MYVGVPTV GEIVELLKTGSAFYAPAASAVAMAQAYLKDSKSVLVCSTYLT-G-QYNVNN-LFVGVPVV AEIVNHLKQGSAFYSPATSVVEMVESIVLDRKRVLTCAVSLD-G-QYGIDG-TFVGVPVK S ATLSMGQAAARFGLSLVRALQGEQGVVECAYVE -G -DGQYARFFSQPLL AEIMKLRGLSSALSAAKAIVDHVHDWMLGTPSGTHVSMAVYSDGNPYGVPGGLIFSFPVT AAVIKARKLSSAMSAAKAICDHVRDIWFGTPEGEFVSMGVISDGNSYGVPDDLLYSFPVV : : : * P falciparum (LDH) P falciprum (MDH) Rickettsia prowazakii Rhizobium laguminosarum Cryptosporidium parvum Chlorobium tepidum E coli Trypanosoma brucei Homo sapiens LGANGVEQVIELQ-LNSEEKAKFDEAIAETKRMKALA -INNKG-AHPVEFP-LTKEEQDLYTESIASVQSNTQKAFDLIK IGKEGVIKVIELQ-LTEEEKILFYKSVTEVKKLIDTIQ IGAGGVERIIEID-LNKTEKEAFDKSVGAVAGLCEACINIAPALK IGKNGIEDVVIVN-LSDDEKSLFSKSVESIQNLVQDLKSLNL LGKNGVEHIYEIK-LDQSDLDLLQKSAKIVDENCKMLDASQG LGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGEEFVNK-CSGGEWQIVSGLN-VTPAISERIKATTTELEEERREVSA -IKNKTWKFVEGLP-INDFSREKMDLTAKELTEEKESAFEFLSSA- 280 273 277 276 275 269 269 289 291 316 313 314 320 316 310 312 327 334 Fig Multiple sequence alignment of MDHs from some representative organisms with deduced amino acid sequence of Pf MDH, for analysis of conserved motifs and amino acids important for enzyme function MDH sequences [P falciparum (AAQ23154.1), Rickettsia 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 (http://www.ebi.ac.uk/clustalw/index.html) using a default parameter The sequence of Pf LDH (Q27743) was also included in the multiple sequence alignment as it shows significant sequence similarity to Pf MDH The N-terminal glycine motif (the nucleotide binding site) and the substrate binding motif have been boxed Other conserved amino acids are indicated by : or * (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 very high concentrations with a Km of 5.44 mM (Table 1) Ó FEBS 2004 3494 A K Tripathi et al (Eur J Biochem 271) Structural characteristics of Pf MDH ldh- P falciparum ldh-P yoelii P.falciparum ldh-MAIZE C.tepidum P.y oelii ldh-A.thaliana R.prowazekii M.loti S.meliloti R.leguminosarum ldh-Human T brucei E.coli Human MOUSE L.major Maize A.thaliana ldh-M.loti T vaginalis ldh-T.vagi nalis ldh-S.meliloti ldh-E.coli 0.1 Fig Phylogenetic tree showing the evolutionary relationship of Pf MDH with various MDHs and LDHs The unrooted tree was constructed by multiple alignments of all the sequences using CLUSTAL X interface with default parameters Misaligned or poorly aligned sequences were manually selected and realigned before constructing the phylogenetic tree 66 45 36 29 24 Fig Overexpression and purification of recombinant Pf MDH E coli cultures were induced by 0.5 mM IPTG for 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, Molecular mass marker proteins; lane 2, soluble extracts 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 and 5, fractions obtained by washing the column with the buffer containing a low concentration (10 mM) of imidazole; lane 6, recombinant Pf MDH protein eluted from a Ni–NTA agarose column in buffer containing 200 mM imidazole; lane 7, second eluate of the column with buffer containing 200 mM inmidazole The model structure of Pf MDH was validated using several tools The Ramachandran plot [38] showed a normal distribution of points with Phi (/) angles mostly restricted 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, Phi for prolines and side chain torsions (v1 and v2) showed no major deviation from the corresponding allowed values PROFILES-3D 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 features of a 2-hydroxyacid dehydrogenase like MDH and LDH including the classical Rossmann fold [39] constituting the NADH binding pocket The structural comparison of some of the relevant 2-hydroxyacid dehydrogenases is provided in Table The overall structure of Pf MDH is quite distinct from that of E coli MDH as evidenced by an rmsd of 5.3 between the Ca coordinates which is not surprising considering the sequence identity of only 26% between the two enzymes On the other hand the structure appears to be similar to that of 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 of the substrate specificity loops (residues 78–94 of Pf MDH) as clearly seen in Fig This is primarily due to the insertion of five residues into the loop in the case of Pf LDH (Fig 1) The substrate specificity loop of Pf MDH shares a sequence identity of 65% with that of the MDH from C tepidum and E coli As expected, the 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 complexed with NADH and OAA (Fig 5) appears to be stabilized by several hydrogen bonds between the enzyme, the substrate and the cofactor in addition to hydrophobic interactions Important residues lining the cofactor binding pocket indicates that Gln11, Asp32, Thr76, Ala77, Val117 and M142 are involved in hydrogen bonding interaction with 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 similar to that in the Pf 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 oxamate except 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 Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3495 OAA reduction 4000 Expression of Pf MDH during asexual intraerythrocytic schizogony 500 Highly synchronized P falciparum cultures were harvested at different developmental stages and expression of Pf MDH was evaluated by analysis of transcripts using Northern blotting (Fig 7A) and the enzyme protein by Western blotting techniques (Fig 7B) MDH in P falciprum 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 trophozoites 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, the polyclonal antibodies against Pf MDH did not cross react with Pf LDH 250 Effect of the inhibitors on Pf MDH and Pf LDH 3000 MDH activity 2000 1000 10 Malate Oxidation 750 10 12 pH Fig Assay of Pf MDH activity at different pH The assay was performed at different pH in phosphate (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 reduced or (NADH oxidized)Ỉmin)1Ỉ(mg protein))1 charge that neutralizes the additional carboxylate group on the substrate and therefore confers substrate specificity to MDH Oxamic acid, which is known to inhibit LDH by interacting with the substrate binding site, inhibited Pf LDH as well as bovine heart LDH with almost the same efficiency (Fig and Table 3) Pf MDH, porcine heart MDH (mitochondrial) and porcine heart MDH (cytosolic) were not inhibited by oxamic acid up to a concentration of mM (Table and Fig 8) Oxamic acid (up to mM) 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 (IC50) of 3.1 lM 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 and Table 3) Gossypol inhibited the growth of P falciparum with an IC50 of 11.5 ± 2.1 lM Berberine, a specific inhibitor of cytosolic as well as organellar MDHs, did not inhibit Pf MDH activity up to a concentration of 10 mM (data not shown) Table Kinetic characterization of recombinant Pf MDH Kinetic characteristics of the recombinant Pf MDH were determined as described in Materials and methods The specific activity is expressed as lmol NADH oxidized or (NAD reduced)Ỉmin)1Ỉmg)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 Reduction (pH 7.5) Oxaloacetate (NADH) NADH (OAA) APADH (OAA) a-Keto-malonate (NADH) Oxidation (pH 10.2) Malate (NAD) NAD (malate) APAD (malate) kcat (1Ỉs)1) Km (mM) 0.030 0.036 0.022 5.44 ± ± ± ± 0.001 0.002 0.001 0.57 1.350 ± 0.024 0.152 ± 0.013 0.370 ± 0.019 kcat/Km (M)1Ỉs)1) 960 950 1010 674 32 26 45 0.12 ± ± ± ± 68 79 150 35 250 ± 19 150 ± 20 175 ± 21 · · · · 106 106 106 106 0.18 · 106 0.98 · 106 0.47 · 106 Specific activity 3657 4870 4935 3899 ± ± ± ± 450 398 238 390 571 ± 38 507 ± 43 562 ± 56 Ó FEBS 2004 3496 A K Tripathi et al (Eur J Biochem 271) Fig Model structure of Pf MDH superimposed on the crystal structure of Pf LDH Pf LDH is shown as a white ribbon, Pf MDH as a yellow ribbon, NADH and oxamate bound to Pf LDH as cyan sticks, NADH and OAA bound to Pf MDH as magenta sticks To highlight the differences the substrate specificity loop is coloured green in the case of Pf LDH and red in the case of Pf MDH Comparative expression of Pf MDH, Pf LDH and Pf MQO and effect of gossypol The effect of gossypol on the expression of Pf LDH and Pf MDH in P falciparum cultures was evaluated Highly synchronized cultures of P falciparum with  15% parasitaemia were treated with 25 and 100 lgỈmL)1 gossypol Table Structural comparison of 2-hydroxyacid dehydrogenases related to Pf MDH Root mean square distance (rmsd) for the Ca atoms is used to compare the protiens’ structures [37] C tepidum E coli T gondii Pf MDHa Pf LDHb MDHc MDHd LDHe Pf MDH Pf LDH C tepidum MDH E coli MDH T gondii LDH a 0.80 0.85 0.80 0.93 0.85 0.93 5.25 3.30 2.50 0.95 0.79 2.11 5.25 0.95 3.30 0.79 2.50 2.11 1.74 1.74 b c d Model structure Dunn et al [14] Dalhus et al [23] Hall and Banaszak [35] e Kavanagh et al [36] and the cultures were harvested after h Control cultures without treatment were set up in parallel Comparative expression of Pf MDH, Pf LDH and Pf MQO were evaluated 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 lgỈmL)1 of gossypol showed significant induction of expression of Pf MDH, while at this concentration expression of Pf LDH and Pf MQO remained unaltered Treatment with 100 lgỈmL)1 gossypol caused further induction of Pf MDH expression while expression of Pf LDH significantly decreased with this treatment (Fig 9A) The Pf MQO expression level remained unchanged upon treatment with 100 mgỈmL)1 gossypol Induction of Pf MDH due to gossypol treatment was further confirmed by Western analysis (Fig 9B) Discussion During asexual intraerythrocytic schizogony, the malaria parasite obtains its energy mainly by utilization of glucose Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3497 Fig Important residues interacting with (A) OAA in Pf MDH and (B) oxamate in Pf LDH [9] The ligands are coloured by atom types whereas the protein residues are shown as yellow sticks Hydrogen bonds are depicted as white dotted lines Hydrogen atoms are not shown for clarity The residues of the Pf LDH structure are numbered in accordance with Fig through glycolysis [1], which 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 of GPI anchors [4] The parasite, however, is equipped with the complete battery of TCA cycle enzymes, although the operation of a mitochondrial TCA cycle and its role in energy generation in malaria parasites is still debatable A recent report has indicated the operation of oxidative phosphorylation in 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 localizations [8] However, the P falciparum genome contains only one full length MDH gene on chromosome 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 demonstrate that this gene is functional in malaria parasites and encodes an NAD+-dependent MDH Another gene (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 (http://www.plamodb.org) This gene, however, seems to be truncated at the N-terminal end Pf LDH also is localized on chromosome 13 of P falciparum The biochemical properties of recombinant Pf MDH are similar to those reported earlier for the enzyme purified from P falciparum extracts [19] The important characteristic of Pf MDH is its high homology with bacterial MDHs and also with LDHs Pf MDH also shows some distinct enzymatic characteristics, i.e strict substrate specificity, insensitivity to inhibition by high concentration of the Ó FEBS 2004 3498 A K Tripathi et al (Eur J Biochem 271) 1.9 kb A 1.39 kb 36.6 KDa B Table Inhibition of P falciparum and porcine MDH and LDH by oxamate and gossypol The enzyme assays were performed with varying concentrations of the inhibitors, at saturating concentrations of the OAA (for MDH) or pyruvate (for LDH) and NADH Values given are mean ± SD of at least three observations 28 KDa ER LR ET LT ES Inhibitor IC50 (lM) LS Fig Expression of Pf MDH in P falciparum cultures during intraerythrocytic 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 to positively charged nylon membrane and hybridized to a radiolabelled Pf MDH probe Membranes were exposed overnight at )80 °C with radiography film to detect the transcripts (B) Western blot analysis The soluble protein lysates prepared from the RBC parasite preparation were separated by SDS/PAGE (10% acrylamide) and transferred to a nitrocellulose membrane The Pf MDH enzyme protein was detected by blotting the membranes with anti-Pf MDH sera (1 : 100) and detection by the peroxidase method ER, Early ring; LR, late ring; ET, early trophozoite; LT, late trophozoite; ES, early schizont; LS, late schizont 120 Gossypol 100 Enzyme activity remaining (%) 80 60 40 20 0.1 120 10 100 1000 Oxamate 100 80 60 40 20 10 100 1000 10000 Inhibitor Concentration (µM) µ Fig Inhibition 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 concentration of pyruvate and NADH, while MDHs were assayed in the presence of saturating concentrations of OAA and NADH For inhibition studies the reaction mixtures containing the enzyme and the inhibitors were preincubated for 10 before initiating 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); porcine heart MDH (cytosolic) (n/solid line) Enzyme Oxamate Gossypol Pf MDH Porcine mitochondrial MDH Porcine cytosolic MDH P falciparum LDH Porcine heart LDH > 2000 > 2000 > 2000 58.0 ± 8.9 78.4 ± 17.9 1.5 2.4 2.9 3.2 84.5 ± ± ± ± ± 0.5 1.1 0.9 0.7 9.5 substrate and utilization of APAD as an alternate cofactor Some cytosolic MDHs show broader substrate specificity and are able to utilize other dicarboxylic keto acids also as the substrates [41] and MDHs are inhibited by high concentrations of OAA [42] Pf MDH was therefore different from these MDHs in its biochemical characteristics 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 malaria diagnostic test, OptimalÒ [43] The MalstatÒ reagent, which is used for selective assay of Pf LDH in P falciparum cultures, is also used for quantification of growth of the malaria parasite in an in vitro antimalarial assay [44,45] Pf LDH utilizes APAD(H) with much higher efficiency than NAD(H), while Pf MDH utilizes APAD(H) and NAD(H) with almost equal efficiency A commercially available NAD+(H) specific MDH from porcine heart (Sigma M 2634) did not utilize APAD(H) even up to 100 mM Pf MDH contains almost all of the amino acids residues, which are typically conserved in cytosolic MDHs [46] R81, R87 and R150, which line the anion binding site of the substrate binding pocket, are positioned to both stabilize and orient the 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 has 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 forms a distant 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 oxygen 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 characteristic 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 Ó FEBS 2004 Malate dehydrogenase of P falciparum (Eur J Biochem 271) 3499 MQO G25 G100 G100 100 Control G100 150 Control LDH 200 G25 MDH Control Expression (% of control) 250 G25 A 50 G25 G100 B MDH Control G25 G100 MDH Expression (% of control) Control LDH 300 MQO G100 250 200 G25 150 100 Control 50 Fig Effect of treatment of P falciparum cultures with gossypol on comparative expression of Pf MDH, Pf LDH and Pf MQO Highly synchronized cultures with approximately 15% parasitaemia were exposed to 25 and 100 lgỈmL)1 gossypol at the early trophozoites stage The cultures were harvested after h of exposure at the late trophozoites stage Expression the enzymes was checked by (A) analysis of equal amounts of RNA by Northern blotting and also by (B) analysis of equal amounts of proteins by Western blotting G25 and G100 indicate the P falciparum cultures treated with 25 and 100 lgỈmL)1 gossypol, respectively P falciparum cultures without treatment (control) were also processed under similar conditions The bars represent relative density of RNA/protein band as compared to controls based on quantitative image analysis 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 regulated in contrast to Pf LDH which is consistently expressed at very high levels throughout the asexual intraerythrocytic development of the malaria parasite [49] Transcription of Pf MDH is initiated during the ring stage, peaking at the early trophozoite stage and decreasing in late schizonts, while the Pf MDH 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 Under normal physiological conditions the expression of Pf MDH is markedly lower than that of Pf LDH Expression of Pf MQO, another enzyme involved in the oxidation of malate and suggested to be localized to mitochondria, was also significantly higher as compared to Pf MDH Recently biochemical evidence has been presented demonstrating the presence of a rotenone insensitive malate : 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 lM while for malate it was quite high (20 mM) Malate oxidation at physiological pH is of interest, but at physiological pH the reaction reaches steady state very soon producing only undetectable changes At pH and 7.5 no oxidation of malate could be detected, even with very high concentration of the enzyme protein and the substrates As protons are the product of this reaction, the free energy difference is highly positive at physiological pH To have the reaction proceed in the direction of oxidation of malate, the ratio of 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 chromosome of P falciparum (http://www.plasmodb.org) MQO catalyses 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 glutamicum (and subsequently reported in several other bacteria including Mycobacterium [52,53] However in the malaria parasite Pf MQO is functional only in combination with 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 of large amounts of haemoglobin by malaria parasite during intraerythrocytic proliferation makes it highly vulnerable to oxidative damage [1] To avoid this oxidative damage, the parasite maintains a low level of oxygen tension Under these conditions oxidation of malate by NAD+ dependent MDH will be highly unfavourable 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 Ó FEBS 2004 3500 A K Tripathi et al (Eur J Biochem 271) Glucose Glycolysis PEP Pathways Pathways Pyruvate Oxaloacetate NADH X LDH Lactate Oxaloacetate MDH + NAD MQO malate malate Cytosol Mitochondria Fig 10 The scheme indicating correlation of Pf LDH, Pf MDH and Pf MQO functions in P falciparum Thickness of the line/arrow indicates relative abundance of the enzyme/enzymatic reaction in the parasite The broken lines/arrows show inhibition and the double lines/ arrows indicate induction of the enzyme/enzymatic reaction in the presence of gossypol the LDH/MDH inhibitor The scheme particularly depicts the role of Pf LDH in NAD/NADH coupling, which may be completed by Pf MDH under the conditions suppression of Pf LDH expression 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 with much lower potency However Pf MDH did not show much selectivity for inhibition by gossypol Both malarial and mammalian MDHs were inhibited 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 almost the same potency Gossypol also inhibits growth of P falciparum in vitro Treatment of a highly synchronized P falciparum cultures (at the early trophozoite stage) with gossypol did not cause any induction 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 the parasite according to its physiological and metabolic requirements; it would be of interest to determine the mechanism of 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 by the malaria parasite under normal physiological circumstances How- ever, induction of expression of Pf MDH caused by treatment with a Pf LDH inhibitor indicates that besides its role in production of malate/OAA for further metabolic reactions, Pf MDH may also complement the Pf LDH function of NAD/NADH coupling reactions and regeneration of NAD+ [14,15,40] In the absence of complete oxidation of glucose by the malaria parasite regeneration of NAD+ is necessary for uninterrupted utilization of glucose and energy generation through glycolysis 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 characteristics of the parasite enzyme, 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 LDH and Pf MDH for complete blockade of energy generation through glucose utilization in 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 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