Purification, characterization and localization of mitochondrial dihydroorotate dehydrogenase in Plasmodium falciparum, human malaria parasite.
The mitochondrial dihydroorotate dehydrogenase (DHODase), the single redox reaction in the pyrimidine de novo synthetic pathway, was purified to near homogeneity by detergent solubilization and fast protein liquid chromatography (FPLC) techniques from the mature trophozoites and schizonts of Plasmodium falciparum, human malaria parasite. The purified DHODase was monofunctional protein with a M(r) of 56,000 +/- 4000, based on Superose 12 gel filtration FPLC and SDS-PAGE analyses. Polyclonal antibodies raised against the purified P. falciparum protein was cross-reacted with P. berghei, rodent malaria parasite. The optimal activity of DHODase required long chain of coenzyme Q (CoQ6-10) which were essential for electron transfer. The Km and kcat values for L-dihydroorotate were 14.4 +/- 5.9 microM and 15.0 +/- 1.4 min-1, respectively; for CoQ6, they were 22.5 +/- 6.4 microM and 21.6 +/- 3.4 min-1. L-Orotate, an enzymatic product, was a strong competitive inhibitor with Ki of 18.2 +/- 3.6 microM. The 5-substituted L-orotates having antimalarial activities against P. falciparum in vitro were found to be competitive inhibitors. The inhibitory effect by these 5-substituted L-orotates on the malarial DHODase was different from the mammalian enzyme. Various benzoquinones and naphthoquinones were found to inhibit the purified DHODase activity at a different degree. Mitochondria from erythrocytic cycle of P. falciparum were purified, using differential 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 what found in many protozoan parasites. DHODase was localized inside the mitochondria as probed by immunogold labeling with the polyclonal antibodies and selective solubilization by digitonin.
Krungkrai
J
J
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001667:Binding, Competitive; D002458:Cell Fractionation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000032:analysis; D006046:Gold; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000032:analysis; D009285:Naphthoquinones / Q000494:pharmacology; D009963:Orotic Acid / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000648:ultrastructure; D014451:Ubiquinone / Q000494:pharmacology
D016227:Benzoquinones; D000090722:Dihydroorotate Dehydrogenase; D009285:Naphthoquinones; D014451:Ubiquinone; D009963:Orotic Acid; D006046:Gold; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV