title: Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei.
abstract: Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].
mesh_terms: D000818:Animals; D003421:Crithidia / Q000201:enzymology*; D004081:Dihydroorotate Oxidase / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D046911:Macromolecular Substances; D010088:Oxidoreductases; D004081:Dihydroorotate Oxidase