Malarial Dihydroorotate Dehydrogenase

Abstract

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidn. of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochem. properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a sol., active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH max. of 8.0 (kcat 8 s-1, Km DHO (40-80 μM)). The substrate specificity of the ubiquinone cofactor (CoQn) that is required for the oxidn. of FMN in the second step of the reaction was also detd. The isoprenoid (n) length of CoQn was a determinant of reaction efficiency; CoQ4, CoQ6 and decylubiquinone (CoQD) were efficiently utilized in the reaction while cofactors lacking an isoprenoid tail (CoQ0 and vitamin K3) showed decreased catalytic efficiency, resulting from a 4 to 7-fold increase in K. Five potent inhibitors of mammalian DHODH (Redoxal, dichloroallyl lawsone or DCL, and three analogs of A77 1726) were tested as inhibitors of the malarial enzyme. All five compds. were poor inhibitors of the malarial enzyme, with IC50 values ranging from 0.1-1.0 mM. The IC50 values for inhibition of the malarial enzyme are 102-104-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents. [on SciFinder(R)]