An α-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum: Cloning and biochemical characterization of the enzyme

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 PfMDH was purified to homogeneity and biochemically characterized as an NAD ⁺(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. PfMDH 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 PfMDH transcripts were detected in trophozoites while the PfMDH protein level remained high in trophozoites as well as schizonts. A highly refined model of PfMDH 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 PfMDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in PfLDH and also in α-proteobacterial MDHs. Oxamic acid did not inhibit PfMDH, while gossypol, which interacts at the nucleotide binding site of oxido-reductases and shows antimalarial activity, inhibited PfMDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of PfMDH, while expression of PfLDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. PfMDH may complement PfLDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of PfMDH and PfLDH may be required to target this pathway and to develop potential new antimalarial drugs.