The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii 
is essential for parasite growth during infection. To investigate the properties 
of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii 
pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD 
exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for 
l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking 
or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As 
expected, fumarate was a poor electron acceptor for this family 2 DHOD. The 
IC(50)s determined for A77-1726, the active derivative of the human DHOD 
inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, 
and 60μM, respectively. The enzyme was not significantly affected by brequinar 
or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known 
inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD 
exhibits a lengthy 157-residue N-terminal extension, consistent with a potential 
organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to 
examine subcellular localization of TgDHOD in transgenic parasites expressing 
the tagged protein. Using both exogenous and endogenous expression strategies, 
anti-myc fluorescence signal colocalized with antibodies against the 
mitochondrial marker ATPase. These findings demonstrate that TgDHOD is 
associated with the parasite's mitochondrion, revealing this organelle as the 
site of orotate production in T. gondii. The TgDHOD gene appears to be essential 
because while gene tagging was successful at the TgDHOD gene locus, attempts to 
delete the TgDHOD gene were not successful in the KU80 background. Collectively, 
our study suggests that TgDHOD is an excellent target for the development of 
anti-Toxoplasma drugs.