Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei.
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].
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*
D046911:Macromolecular Substances; D010088:Oxidoreductases; D004081:Dihydroorotate Oxidase