Isocitrate dehydrogenase of Plasmodium falciparum.
Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+-dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 micro m and 22 micro m, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 micro m and that of d-isocitrate was determined to be 40 micro m. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites.
Wrenger
Carsten
C
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, UK.
Müller
Sylke
S
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004734:Energy Metabolism; D004912:Erythrocytes / Q000469:parasitology; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013552:Swine
D017931:DNA Primers; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)