Glutamate dehydrogenase, the marker protein of Plasmodium falciparum--cloning, expression and characterization of the malarial enzyme.
The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern-blot analysis indicates a single-copy gene. The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.
Wagner
J T
JT
Biochemie-Zentrum der Universität Heidelberg, Germany.
Lüdemann
H
H
Färber
P M
PM
Lottspeich
F
F
Krauth-Siegel
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers / Q000737:chemistry*; D002738:Chloroquine / Q000494:pharmacology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000382:microbiology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D005969:Glutamate Dehydrogenase / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D015415:Biomarkers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002738:Chloroquine; D005969:Glutamate Dehydrogenase