Functional characterization of an alternative [lactate dehydrogenase-like] malate dehydrogenase in Plasmodium falciparum

Chan, Maurice; Sim, Timothy

doi:10.1007/s00436-003-0996-1

Cited by 24 publications

(11 citation statements)

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“…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).…”

Section: Discussionsupporting

confidence: 84%

An α‐proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum

Tripathi

Desai²,

Pradhan

et al. 2004

European Journal of Biochemistry

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Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD + (H)-specific MDH, which catalysed reversible 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.Keywords: gossypol; lactate dehydrogenase; malate dehydrogenase; malate:quinone oxidoreductase; Plasmodium falciparum.The enzymes associated with carbohydrate and energy metabolism in malarial parasites have attracted significant attention as potential targets for new antimalarial drug discovery [1]. During asexual reproduction and growth within the host's erythrocytes the parasite depends mainly on the glycolytic pathway to obtain energy. Infected 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-MeyerhofParnas 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 ...

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Section: Discussionsupporting

confidence: 84%

An α‐proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum

Tripathi

Desai²,

Pradhan

et al. 2004

European Journal of Biochemistry

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“…For vaccine development, Cs-mMDH has 65% sequence similarity with human mMDH; further immunological research will be done to confirm whether the antibody induced by Cs-mMDH has cross-reacted with human mMDH. The superimposition of the model obtained for MDH of Plasmodium falciparum over porcine cytosolic MDH revealed significant conformational differences in the active site loop and other solvent-exposed regions; these observation suggested a large scope for the use of some of the domains of malarial MDHs as potential drug targets (Chan and Sim 2004). Further efforts will be made to obtain crystals of Cs-mMDH and determine threedimensional structure by X-ray crystallography.…”

Section: Discussionmentioning

confidence: 98%

“…The glycolytic enzymes including mMDH are recognized as crucial molecules for trematode survival and have been targeted for vaccine development (Hong et al 2000) and for drug screening (Chan and Sim 2004). On the other hand, tracing the history of MDH isoenzymes in eukaryotic evolution is handicapped by the lack of sequences from early branching eukaryotes (Sogin 1991).…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis

Zheng¹,

Huang²,

et al. 2008

Parasitol Res

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The NAD-dependent mitochondrial malate dehydrogenase (mMDH, EC1.1.1.37) plays pivotal roles in tricarboxylic acid and is crucial for the survival and pathogenecity of parasites. A cDNA, which was identified by high throughput sequencing from the cDNA library constructed from adult Clonorchis sinensis, encoded a putative peptide of 341 amino acids with more than 50% identity with mMDHs from other organisms. The mMDH was expressed in Escherichia coli as the recombinant protein with a GST tag and purified by glutathione-Sepharose 4B column. The recombinant mMDH showed MDH activity of 63.6 U/mg, without lactate dehydrogenase activity and NADPH selectivity. The kinetic constants of recombinant mMDH were determined.

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“…The completion of the Plasmodium genome project also revealed genes encoding subunits of the ATP synthase and all enzymes of the TCA cycle (Gardner et al ., 2002), despite previous failures to detect them. Several TCA cycle components appear to be functional (Suraveratum et al ., 2000;Wrenger and Muller, 2003;Chan and Sim, 2004). It remains to be seen whether these enzymes are in fact mitochondrially targeted.…”

Section: Discussionmentioning

confidence: 99%

Strange organelles –Plasmodium mitochondria lack a pyruvate dehydrogenase complex

Ralph

2004

Molecular Microbiology

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SummaryOur understanding of the Plasmodium mitochondrion and apicoplast has been greatly assisted by the genome sequence project. Sequence data have seeded recent research showing that the apicoplast is the site of several anabolic pathways including fatty acid synthesis. The discovery of an active apicoplast pyruvate dehydrogenase complex implies this enzyme generates the acetyl-CoA needed for fatty acid synthesis. However, the absence of a corresponding mitochondrial complex suggests that energy generation in Plasmodium is considerably different from pathways described in other eukaryotes.Eight years ago it was shown that apicomplexans including Plasmodium , the causative agent of malaria, possessed a plastid (later known as the apicoplast). Apicomplexans are non-photosynthetic, so the much-asked question was why keep a plastid? The riddle is by no means solved, but bioinformatic analyses, enzyme biochemistry and inhibitor studies all point to dependence on the plastid for anabolic pathways whose cytosolic counterparts were lost after the plastid was acquired. The first such pathway discovered in Plasmodium falciparum and Toxoplasma gondii was fatty acid biosynthesis, a pathway that shows much potential as a chemotherapeutic target (Surolia and Surolia, 2001;Waller et al ., 2003). In this issue of Molecular Microbiology , two articles (by Foth et al . and by McMillan et al . respectively) describe the characterization of a key enzyme at the starting point of this pathway, the multiprotein complex pyruvate dehydrogenase (PDH). The authors discovered that each of the four subunits required to form PDH contained an Nterminal apicoplast-like targeting sequence, suggesting post-translational trafficking. GFP fusion constructs made for three of these four subunits confirmed that their leaders are sufficient to mediate apicoplast targeting. A second isoform of the E3 (or dihydrolipoamide dehydrogenase) subunit targets to the mitochondria, where it is probably required by branched chain a -keto acid dehydrogenase, a -ketoglutarate dehydrogenase or the glycine decarboxylase complex.The demonstration that the apicoplast contains a PDH suggests that the carbon (and some reducing power) required for apicoplast fatty acid synthesis comes from the conversion of pyruvate to acetyl-CoA by PDH. This fits nicely with the recent consensus that pyruvate is the most important carbon source for fatty acids in plant plastids. However, in clearing up one metabolic conundrum the authors pose another: if the single PDH in Plasmodium belongs to the plastid, where is the mitochondrial PDH? The authors performed a rigorous screen for PDH subunits from available data for six Plasmodium species and for T. gondii -with the same results for all speciesabundant PDH subunits with apicoplast leaders, but only E3 subunits for the mitochondria. Several mitochondrial complexes generally share E3, so the evidence for the absence of mitochondrial PDH in Plasmodium is strong.Like plastids, mitochondria result from the endosymbiosis of a eubacterial ...

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Functional characterization of an alternative [lactate dehydrogenase-like] malate dehydrogenase in Plasmodium falciparum

Cited by 24 publications

References 11 publications

An α‐proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum

An α‐proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum

Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis

Strange organelles –Plasmodium mitochondria lack a pyruvate dehydrogenase complex

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