Molecular biology and biochemistry of malarial parasite pyrimidine biosynthetic pathway.

Metabolic pathways in the malarial parasite are markedly different from the host, eg, hemoglobin, fatty acids, folate and nucleic acids. Understanding of metabolic function will illuminate new chemotherapeutic targets for drug development, including the identification of target(s) for drugs in current use. The parasite-contained pyrimidine biosynthetic pathway is essential for growth and development in the human host. Plasmodium falciparum carbonic anhydrase, producing HCO3- as a pyrimidine precursor, was identified as alpha- type and the encoded gene was cloned and sequenced. The first six enzymes, catalyzing the conversion of HCO3-, ATP, L-aspartate and L-glutamine to uridine 5'-monophosphate (UMP), were partially characterized. The genes encoding these enzymes were identified in order, from the first to the sixth step, as CPSII (carbamyl phosphate synthase II), ATC (aspartate transcarbamylase), DHO (dihydroorotase), DHOD (dihydroorotate dehydrogenase, DHOD), OPRT (orotate phosphoribosyltransferase, OPRT), and OMPDC (orotidine 5'-monophosphate decarboxylase, OMPDC). Unlike its analogous parasitic protozoan, Trypanosoma, the organization of the malarial genes was not an operon-like cluster. The CPSII, DHO and OPRT genes were conserved to bacterial counterparts, whereas the ATC, DHOD and OMPDC were mosaic variations. The data support the mosaic pyrimidine pathway in the malarial parasite. The human host had five enzymes out of the six associated into two different multifunctional proteins, in that a single gene CPSII-ATC-DHO encoded the first three enzymes, and another gene OPRT-OMPDC encoded the last two enzymes. In the malarial parasite, the CPSII and ATC were not characterized. The DHO was partially characterized in Plasmodium berghei. The DHOD was well characterized in both P. falciparum and P. berghei. It was functionally expressed in Escherichia coli. The physical and kinetic properties of the recombinant pfDHOD were similar to the native enzyme. The OPRT and OMPDC were also partially characterized. These lines of evidence indicate that the malarial pyrimidine enzymes are mono-functional forms. In addition, the enzymatic activities inter-converting uracil, uridine and UMP of the pyrimidine salvage pathway, were demonstrated, and the gene encoding uridine phosphorylase was cloned. Our results suggest that the pyrimidine enzymes are possible new drug targets.