Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei

• Published in: Biochemistry (Easton) Vol. 30; no. 7; pp. 1934 - 1939
• Main Authors: Krungkrai, Jerapan, Cerami, Anthony, Henderson, Graeme B
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 19-02-1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Cell Membrane - enzymology; Chromatography, Affinity; Chromatography, Gel; Detergents; Dihydroorotate Oxidase - isolation & purification; Dihydroorotate Oxidase - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Iron - analysis; Kinetics; Molecular Weight; Octoxynol; Oxidoreductases; Plasmodium berghei; Plasmodium berghei - enzymology; Polyethylene Glycols; Sulfur - analysis; Protozoa; Plasmodium berghei; Purification; Radiolabelling; Affinity chromatography; Gel electrophoresis; Iron; Metalloenzyme; Sporozoa; Cofactor; Dihydroorotate oxidase; Immunological method
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00221a029

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].