An alpha-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 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 alpha-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.
National Center for Natural Product Research, Research Institute of Pharmacological Sciences, University of Mississippi, MS 38677, USA.
Abhai K
AK
Tripathi
Prashant V
PV
Desai
Anupam
A
Pradhan
Shabana I
SI
Khan
Mitchell A
MA
Avery
Larry A
LA
Walker
Babu L
BL
Tekwani
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D006072:Gossypol / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010072:Oxamic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; C471971:malate dehydrogenase, FAD-dependent; D006072:Gossypol; D010072:Oxamic Acid