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.