Human
malaria parasite, Plasmodium falciparum, can only synthesize
pyrimidine nucleotides using the de novo pathway, whereas mammalian cells obtain
pyrimidine nucleotides from both the de novo and salvage pathways. The parasite's
orotate phosphoribosyltransferase (PfOPRT) and
orotidine 5'-monophosphate decarboxylase (PfOMPDC) of the de novo
pyrimidine pathway are attractive targets for
antimalarial drug development. Previously, we have reported that the two
enzymes in P. falciparum exist as a multienzyme complex containing two subunits each of 33-kDa PfOPRT and 38-kDa PfOMPDC. In this report, the gene encoding PfOPRT has been cloned and expressed in Escherichia coli. An open reading frame of PfOMPDC gene was identified in the
malaria genome database, and PfOMPDC was cloned from P. falciparum
cDNA, functionally expressed in E. coli, purified, and characterized. The
protein sequence has <20% identity with human OMPDC and four microbial OMPDC for which crystal structures are known. Recombinant PfOMPDC was catalytically active in a dimeric form. Both recombinant PfOPRT and PfOMPDC monofunctional
enzymes were kinetically different from the native multienzyme complex purified from P. falciparum. Oligomerization of PfOPRT and PfOMPDC cross-linked by
dimethyl suberimidate indicated that they were tightly associated as the heterotetrameric 140-kDa complex, (PfOPRT)2(PfOMPDC)2. Kinetic analysis of the PfOPRT-PfOMPDC associated complex was similar to that of the native P. falciparum
enzymes and was different from that of the bifunctional human
enzymes. Interestingly, a nanomolar inhibitor of the yeast OMPDC, 6-thiocarboxamido-uridine 5'-monophosphate, was about 5 orders of magnitude less effective on the PfOMPDC than on the yeast
enzyme. Our results support that the
malaria parasite has unique structural and functional properties, sharing characteristics of the monofunctional
pyrimidine-metabolizing
enzymes in prokaryotes and bifunctional complexes in eukaryotes.