Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo 
pyrimidine biosynthesis and is a proven drug target for inducing 
immunosuppression in therapy of human disease as well as a rapidly emerging drug 
target for treatment of malaria. In Toxoplasma gondii, disruption of the first, 
fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil 
auxotrophy. However, previous attempts to generate uracil auxotrophy by 
genetically deleting the mitochondrion-associated DHODH of T. gondii (TgDHODH) 
failed. To further address the essentiality of TgDHODH, mutant gene alleles 
deficient in TgDHODH activity were designed to ablate the enzyme activity. 
Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene 
alleles induced uracil auxotrophy. Catalytically deficient TgDHODH localized to 
the mitochondria, and parasites retained mitochondrial membrane potential. These 
results show that TgDHODH is essential for the synthesis of pyrimidines and 
suggest that TgDHODH is required for a second essential function independent of 
its role in pyrimidine biosynthesis.