Malarial dihydroorotate dehydrogenase: Substrate and inhibitor specificity

Jeffrey Baldwin, Azizeh M. Farajallah, Nicholas A. Malmquist, Pradipsinh K. Rathod, Margaret A. Phillips

Research output: Contribution to journalArticle

91 Citations (Scopus)

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 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 (kcat 8 s-1, Km app DHO (40-80 μM)). The substrate specificity of the ubiquinone cofactor (CoQn) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQn was a determinant of reaction efficiency; CoQ4, CoQ6 and decylubiquinone (CoQD) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ0 and vitamin K3) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in Km 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 IC50's 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.

Original languageEnglish (US)
Pages (from-to)41827-41834
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number44
DOIs
StatePublished - Nov 1 2002

Fingerprint

Substrate Specificity
Enzyme Inhibitors
Substrates
Enzymes
Flavin Mononucleotide
Terpenes
dichloroallyl lawsone
A 771726
Inhibitory Concentration 50
Biosynthesis
Parasites
Vitamin K 3
Pyrimidines
Ubiquinone
Antimalarials
Oxidation
Mitochondria
dihydroorotate dehydrogenase
Tail
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Malarial dihydroorotate dehydrogenase : Substrate and inhibitor specificity. / Baldwin, Jeffrey; Farajallah, Azizeh M.; Malmquist, Nicholas A.; Rathod, Pradipsinh K.; Phillips, Margaret A.

In: Journal of Biological Chemistry, Vol. 277, No. 44, 01.11.2002, p. 41827-41834.

Research output: Contribution to journalArticle

Baldwin, Jeffrey ; Farajallah, Azizeh M. ; Malmquist, Nicholas A. ; Rathod, Pradipsinh K. ; Phillips, Margaret A. / Malarial dihydroorotate dehydrogenase : Substrate and inhibitor specificity. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 44. pp. 41827-41834.
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AU - Phillips, Margaret A.

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AB - 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 (kcat 8 s-1, Km app DHO (40-80 μM)). The substrate specificity of the ubiquinone cofactor (CoQn) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQn was a determinant of reaction efficiency; CoQ4, CoQ6 and decylubiquinone (CoQD) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ0 and vitamin K3) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in Km 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 IC50's 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.

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