Plasmodium falciparum, the causative agent of the most lethal form of human 
malaria, relies on de novo pyrimidine biosynthesis. A gene encoding orotate 
phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway 
catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate 
(PP(i)) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified 
from P. falciparum (pfOPRT). The deduced amino acid sequence for pfOPRT was 
compared with OPRTs from other organisms and found to be most similar to that of 
Escherichia coli. The catalytic residues and consensus sequences for substrate 
binding in the enzyme were conserved among other organisms. The pfOPRT was 
exceptional in that it contained a unique insertion of 20 amino acids and an 
amino-terminal extension of 66 amino acids, making the longest amino acid 
sequence (281 amino acids with a predicted molecular mass of 33kDa). The cDNA of 
the pfOPRT gene was cloned, sequenced and functionally expressed in soluble 
form. The recombinant pfOPRT was purified from the E. coli lysate by two steps, 
nickel metal-affinity and gel-filtration chromatography. From 1l E. coli 
culture, 1.2-1.5mg of pure pfOPRT was obtained. SDS-PAGE revealed that the 
pfOPRT had a molecular mass of 33kDa and analytical gel-filtration 
chromatography showed that the enzyme activity eluted at approximately 67kDa. 
Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it 
was confirmed that the native enzyme exists in a dimeric form. The steady state 
kinetics of initial velocity and product inhibition studies indicate that the 
enzyme pfOPRT follows a random sequential kinetic mechanism. Compounds aimed at 
the pfOPRT nexus may act against the parasite through at least two mechanisms: 
by directly inhibiting the enzyme activity, or be processed to an inhibitor of 
thymidylate synthase. This study provides a working system with which to 
investigate new antimalarial agents targeted against P. falciparum OPRT.