Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man

Plasmepsins are aspartic proteinases of the malaria parasite, and seven groups of plasmepsins have been identified by comparing genomic sequence data available for the genes encoding these enzymes from Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium berghei, and Plasmodium yoelii. The food vacuole plasmepsins typified by plasmepsin 4 from P. falciparum (PfPM4) constitute one of these groups. Genes encoding the ortholog of PfPM4 have been cloned from Plasmodium ovale, Plasmodium malariae, and P. vivax. In addition, P. falciparum contains three paralagous food vacuole plasmepsins or plasmepsin-like enzymes that appear to have arisen by gene duplication, plasmepsins 1 (PfPM1), 2 (PfPM2) and HAP, and all four were localized to purified food vacuole preparations by two-dimensional gel electrophoresis and mass spectroscopic analysis. The three paralogs of PfPM4 do not have counterparts in the six other Plasmodium spp. examined by genomic DNA blot analysis and by review of available genomic sequence data. The presence of these paralogs among the food vacuole plasmepsins in P. falciparum as compared with the other three species causing malaria in man will impact efforts to rationally design antimalarials targeting the food vacuole plasmepsins.     

Engagement of the S1, S1' and S2' subsites drives efficient catalysis of peptide bond hydrolysis by the M1-family aminopeptidase from Plasmodium falciparum

The M1-family aminopeptidase PfA-M1 catalyzes the last step in the catabolism of human hemoglobin to amino acids in the Plasmodium falciparum food vacuole. In this study, the structural features of the substrate that promote efficient PfA-M1-catalyzed peptide bond hydrolysis were analyzed. X-Ala and Ala-X dipeptide substrates were employed to characterize the specificities of the enzyme's S1 and S1' subsites. Both subsites exhibited a preference for basic and hydrophobic sidechains over polar and acidic sidechains. The relative specificity of the S1 subsite was similar over the pH range 5.5-7.5. Substrate P1 and P1' residues affected both K(m) and k(cat), revealing that sidechain-subsite interactions not only drive the formation of the Michaelis complex but also influence the rates of ensuing chemical steps. Only a small fraction of the available binding energy was exploited in interactions between substrate sidechains and the S1 and S1' subsites, which indicates a modest level of complementarity. There was no correlation between S1 and S1' specificities and amino acid abundance in hemoglobin. Interactions between PfA-M1 and the backbone atoms of the P1' and P2' residues as well as the P2' sidechain further contributed to the catalytic efficiency of substrate hydrolysis. By demonstrating the engagement of multiple, broad-specificity subsites in PfA-M1, these studies provide insight into how this enzyme is able to efficiently generate amino acids from highly sequence-diverse di- and oligopeptides in the food vacuole.     

Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum

Aspartic proteases participate in a wide variety of cellular processes in eukaryotic organisms. The genome of the human malaria parasite Plasmodium falciparum encodes 10 aspartic protease homologs. Functions have been assigned to four of these: plasmepsins I, II, IV and histo-aspartic protease are key players in the catabolism of hemoglobin in the food vacuole. The functions of the other six remain obscure. To better understand the roles of aspartic proteases in blood stage growth and asexual reproduction of P. falciparum, we have characterized the biosynthesis, cellular location and pepstatin-binding properties of plasmepsin V (PM V). PM V is expressed over the course of asexual intraerythrocytic development. The amount of PM V in the parasite is lowest in the ring stage and increases steadily through schizogony. The proregion of this aspartic protease homolog exhibits remarkable interspecies diversity and appears not to be removed following biosynthesis. In intraerythrocytic parasites, PM V is located in the endoplasmic reticulum but not in ERD2-associated Golgi structures. Fractionation and solubilization experiments demonstrate that PM V is an integral membrane protein, a result that is consistent with the presence of a C-terminal putative transmembrane domain in the PM V sequence. In contrast to the food vacuole plasmepsins, detergent-solubilized PM V does not bind the aspartic protease inhibitor pepstatin. Together, these results strongly suggest that the role of PM V in P. falciparum is distinct from those of previously characterized plasmepsins.     

Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax.

The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli. The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined. Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme. The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were approximately 60 to >4000 times less active against the mutant enzymes. In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred approximately 4000- and approximately 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR. The S58R+S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Km value than the single mutant. The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants. Data on kinetic parameters and inhibitory constant suggest that the wild-type P. vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P. vivax are responsible for antifolate resistance.     

Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus

As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.     

Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria

Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Δpm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.The digested vacuole (DV) of malaria parasites performs hemoglobin degradation, which is a crucial process for parasite growth and survival within the host erythrocyte. In Plasmodium falciparum, the most important human malaria parasite, this is achieved with the contribution of several digestive vacuole proteases including three aspartic proteinases, the plasmepsins (PM) PfPM1, PfPM2, and PfPM4 and one histo-aspartic protease, PfHAP.^1–5 The plasmepsins have long been studied as potential drug targets and subjected to functional and biochemical studies with the hope that inhibiting them would halt hemoglobin digestion and result in parasite death. Surprisingly, the systematic disruption of either individual or different combinations of the plasmepsin genes did not result in any striking growth defect. Presumably, this is due to redundant enzyme systems for digesting hemoglobin, which involve cysteine proteases, metalloproteases, and aminopeptidases, and to the presence of multiple pathways for the uptake of extracellular amino acids.^6–8 The P. falciparum and Plasmodium reichnowi clades differ from other Plasmodium species in that they have four genes encoding DV plasmepsins. In P. falciparum only the disruption of all four plasmepsin genes, which eliminates all aspartic protease activity from the DV, resulted in delayed in vitro schizont maturation accompanied by reduced formation of hemozoin (an insoluble crystal produced during hemoglobin degradation) and less efficient processing of endosomal vesicles in the DV.⁴We here investigated the impact of the loss of the various functions of the DV plasmepsins on parasite virulence by disrupting the single gene encoding the DV plasmepsin 4 (pm4) in the rodent malaria parasite Plasmodium berghei. This parasite is a well established and tractable model to study the function of Plasmodium genes in vivo and replicates several key features of human cerebral malaria.^9,10 The phenotypic analysis of loss-of-function mutants has been used to gain an insight into a variety of host-parasite interactions.¹¹ In this study, we confirm that the disruption of PM4, which results in loss of all aspartic proteinase activity targeted to its lysosomal compartments, has only a modest effect on the intraerythrocytic development of P. berghei parasites, but we observed dramatic differences in the virulence of these parasites compared with that of wild-type parasites. Specifically, we report the growth and multiplication characteristics of Δpm4 parasites in different mouse strains and demonstrate that these parasites neither induce experimental cerebral malaria (ECM) in ECM-susceptible mice nor kill the host by hemolytic anemia in ECM-resistant mice. In these latter mice, Δpm4 parasites induce a self-resolving infection, which generates spleen-dependent protective immune responses. This is the first report of a mutant P. berghei parasite that does not induce cerebral complications as the result of a single gene mutation.     

Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum

Intraerythrocytic Plasmodium falciparum digests vast amounts of hemoglobin within an acidic food vacuole (FV). Four homologous aspartic proteases participate in hemoglobin degradation within the FV. Plasmepsin (PM) I and II are thought to initiate degradation of the native hemoglobin molecule. PM IV and histo-aspartic protease (HAP) act on denatured globin further downstream in the pathway. PM I and II have been shown to be synthesized as zymogens and activated by proteolytic removal of a propiece. In this study, we have determined that the proteolytic processing of FV plasmepsins occurs immediately after a conserved Leu-Gly dipeptidyl motif with uniform kinetics and pH and inhibitor sensitivities. We have developed a cell-free in vitro processing assay that generates correctly processed plasmepsins. Our data suggest that proplasmepsin processing is not autocatalytic, but rather is mediated by a separate processing enzyme. This convertase requires acidic conditions and is blocked only by the calpain inhibitors, suggesting that it may be an atypical calpain-like protease.     

Purification and biochemical characterization of a novel glutamyl endopeptidase secreted by a clinical isolate of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a ubiquitous Gram-positive pathogenic bacterium responsible for a majority of skin infections and toxic shock syndromes. In this study, a 34-kDa glutamate-specific serine protease (named VSPase) secreted by a clinical isolate of S. aureus sp. strain C-66 was purified and characterized, and VSPase-encoding gene was also cloned by PCR. VSPase enzyme purified from culture supernatant and its recombinant enzyme expressed in E. coli exhibited a proteolytic activity over a broad range of pH (6.0-8.5) and showed an optimal activity at 45 ?C. The enzyme activity was completely inhibited by DFP. The N-terminal sequence of native VSPase showed that the enzyme was produced as a form of zymogen and activated to a functional enzyme by losing its N-terminal 68 amino acid residues. VSPase specifically cleaved peptide bonds at the carboxyl sides of glutamate residues in a protein substrate such as prothrombin and exhibited its amidolytic activity towards a chromogenic substrate, Z-Phe-Leu-Glu-pNA (L-2135). The Km, kcat and kcat/Km values for VSPase were estimated to be 1.48 ± 0.156 mM, 44.4 ± 2.66/sec and 30/mM/sec, respectively, when L-2135 was used as a substrate. It was revealed by site-directed mutagenesis that one of substitution mutations resulted in His119, Asp161 and Ser237 residues of VSPase abolished the enzyme activity dramatically, suggesting that the three amino acid residues may compose a catalytic triad in VSPase as in typical serine proteases. Taken together, the results obtained by the present study demonstrate that VSPase is a typical glutamate-specific serine endopeptidase.     

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.     

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.     

Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci)

The multifunctional enzyme thioredoxin-glutathione reductase (TGR) was purified to homogeneity from the soluble fraction of Taenia crassiceps metacestode (cysticerci). Specific activities of 17.5 and 4.7 U mg(-1) were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3 microM were respectively calculated for thioredoxin, GSSG and NADPH. The kcat/Km ratio of T. crassiceps TGR for both thioredoxin and GSSG falls in the range observed for typical thioredoxin reductases and glutathione reductases. Purified enzyme also showed glutaredoxin activity, with a specific activity of 19.2 U mg(-1) with hydroxyethyl disulfide as substrate. Both thioredoxin and GSSG disulfide reductase activities were fully inhibited by nanomolar concentrations of the gold compound auranofin, supporting the existence of an essential selenocysteine residue. Relative molecular mass of native enzyme was 136,000 +/- 3000, while the corresponding value per subunit, obtained under denaturing conditions, was 66,000 +/- 1000. These results suggest TGR exists as a dimeric protein. Isoelectric point of the enzyme was at pH 5.2. Moderate or high concentrations of GSSG, but neither thioredoxin nor NADPH, resulted in a markedly hysteretic kinetic, characterized by a lag time before the steady state velocity was reached. The magnitude of the lag time was dependent on GSSG and enzyme concentration. Preincubation of the enzyme with micromolar concentrations of GSH or DTT abolished the hysteresis, suggesting that a thiol-disulfide exchange mechanism is involved.     

Activation of human rhinovirus-14 3C protease

The catalytic efficiency of human rhinovirus-14 (HRV14) 3C protease as a function of solvents and other regulators has been investigated using synthetic peptides as substrates. The proteolytic activity of HRV14 3C was found to be strongly stimulated by a series of anions in vitro and the activation was accompanied by changed Km, kcat, and increased kcat/Km values. A more than 72-fold increase in the 3C catalytic efficiency toward peptide substrates was observed in the presence of 0.8 M sodium sulfate. Several approaches, including size-exclusion chromatography and chemical cross-linking experiments, suggested that no oligomerization of the 3C enzyme occurred in the presence of activating anions. However, the anions did induce a significant conformational change of HRV14 3C protease, as revealed by circular dichroism spectrometry and tyrosine fluorescence analyses, which might contribute to 3C enzyme activation. Finally, the results obtained from 3C protease inhibitor studies suggested that the S1 specificity pocket of HRV14 3C was mainly affected by the activating anions. An induced-fit catalysis mechanism for viral proteases is discussed.     

The extended cleavage specificity of the rodent beta-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase

In rat and mouse the phylogenetic homologues of the human mast cell alpha-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate alpha-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell beta-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1' and P3'. The two enzymes differ clearly only in one position, the P2' residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2' of known substrates for the human chymase. Overall, these two rodent beta-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.     

An algorithm to predict 3' intron splice sites in Plasmodium falciparum genomic sequences

A new algorithm, PfAGSS, for predicting 3' splice sites in Plasmodium falciparum genomic sequences is described. Application of this program to the published P. falciparum chromosome 2 and 3 data suggests that existing programs result in a high error rate in assigning 3' intron boundaries.     

Analysis of the sequences flanking the translational start sites of Plasmodium falciparum

The 5' and 3' regions adjacent to the initiation codon in 22 Plasmodium falciparum sequences were examined. A 5' consensus sequence (AAAA/ATG) was found. Although P. falciparum non-translated DNA is A-rich, A occurred significantly more frequently in the 4 positions preceding the initiation ATG than in adjacent non-translated DNA, suggesting that this consensus sequence has functional significance in the initiation of translation. This region has similarities with the equivalent sequences in yeast and Drosophila but differs markedly from that in vertebrates. No significant bias in nucleotide frequencies was found 3' to the initiation codon.     

Purification and characterization of Plasmodium falciparum succinate dehydrogenase

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.     

Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum

Erythrocytic stages of the malaria parasite Plasmodium falciparum express four related papain-family cysteine proteases, termed falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize biological roles, we attempted disruption of each falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1, falcipain-2, and falcipain-2' was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2' knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results, falcipain-2 knockout trophozoites developed swollen, hemoglobin-filled food vacuoles, indicative of a block in hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to cysteine protease inhibitors and over 1000 times more sensitive to an aspartic protease inhibitor. The falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for falcipain-2 and falcipain-3 in the development of erythrocytic malaria parasites and a complex interplay between P. falciparum cysteine and aspartic proteases.     

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.     

Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase

Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph chi2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS k(cat) value of the R470D+C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Km values for dUMP and CH2H4folate of the R470D+C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D+C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS.