Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo 
pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the 
parasitic protist causing Chagas' disease. T. cruzi and human DHOD have 
different biochemical properties, including the electron acceptor capacities and 
cellular localization, suggesting that T. cruzi DHOD may be a potential 
chemotherapeutic target against Chagas' disease. Here, we report nucleotide 
sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the 
recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 
and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 
and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb 
DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, 
and encode proteins of 314 amino acids. The three DHOD genes differ by 26 
nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all 
residues critical for constituting the active site are conserved among the three 
proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess 
similar enzymatic properties, including optimal pH, optimal temperature, Vmax, 
and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and 
Km for both substrates. Orotate competitively inhibited all three DHOD enzymes 
to a comparable level. These results suggest that, despite their genetic 
variations, kinetic properties of the three T. cruziDHODs are conserved. Our 
findings facilitate further exploitation of T. cruzi DHOD inhibitors, as 
chemotherapeutic agents against Chagas' disease.