Malarial dihydroorotate dehydrogenase. Substrate and inhibitor specificity.
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 oxidation 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 biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm. The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-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.
Baldwin
Jeffrey
J
Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, 75390-9041, USA.
Farajallah
Azizeh M
AM
Malmquist
Nicholas A
NA
Rathod
Pradipsinh K
PK
Phillips
Margaret A
MA
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001713:Biphenyl Compounds / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D003437:Crotonates; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism; D008969:Molecular Sequence Data; D009570:Nitriles; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010963:Plasmodium falciparum / Q000201:enzymology*; D013379:Substrate Specificity; D014052:Toluidines; D014451:Ubiquinone / Q000378:metabolism; D011798:Quinaldines
D000814:Aniline Compounds; D001713:Biphenyl Compounds; D003437:Crotonates; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D006885:Hydroxybutyrates; D009570:Nitriles; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D014052:Toluidines; D014451:Ubiquinone; C527525:teriflunomide; C046943:brequinar; D011798:Quinaldines