PURIFICATION, CHARACTERIZATION AND LOCALIZATION OF MITOCHONDRIAL DIHYDROOROTATE DEHYDROGENASE IN PLASMODIUM-FALCIPARUM, HUMAN MALARIA PARASITE

The mitochondrial dihydroorotate dehydrogenase (DHODase), the single r edox reaction in the pyrimidine de novo synthetic pathway, was purifie d to near homogeneity by detergent solubilization and fast protein liq uid chromatography (FPLC) techniques from the mature trophozoites and schizonts of Plasmodium falciparum, human malaria parasite. The purifi ed DHODase was monofunctional protein with a M(r) of 56000 +/- 4000, b ased on Superose 12 gel filtration FPLC and SDS-PAGE analyses. Polyclo nal antibodies raised against the purified P. falciparum protein was c ross-reacted with P. berghei, rodent malaria parasite. The optimal act ivity of DHODase required long chain of coenzyme Q (CoQ(6-10)) which w ere essential for electron transfer. The K-m and k(cat) values for L-d ihydroorotate were 14.4 +/- 5.9 mu M and 15.0 +/- 1.4 min(-1), respect ively; for CoQ(6), they were 22.5 +/- 6.4 +/- mu M and 21.6 +/- 3.4 mi n(-1). L-Orotate, an enzymatic product, was a strong competitive inhib itor with K-i of 18.2 +/- 3.6 mu M. The 5-substituted L-orotates havin g antimalarial activities against P. falciparum in vitro were found to be competitive inhibitors. The inhibitory effect by these 5-substitut ed L-orotates on the malarial DHODase was different from the mammalian enzyme. Various benzoquinones and naphthoquinones were found to inhib it the purified DHODase activity at a different degree. Mitochondria f rom erythrocytic cycle of P. falciparum were purified, using different ial centrifugation and followed by Percoll density gradient separation , with purifications of 13-fold and overall yields of 33%. The double- membraned mitochondria had a few tubular-like cristae structure as wha t found in many protozoan parasites. DHODase was localized inside the mitochondria as probed by immunogold labeling with the polyclonal anti bodies and selective solubilization by digitonin.