A Novel Enzyme Complex of Orotate Phosphoribosyltransferase and Orotidine 5‘-Monophosphate Decarboxylase in Human Malaria Parasite Plasmodium falciparum:  Physical Association, Kinetics, and Inhibition Characterization^,

Abstract

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.

Identification and Validation of Novel Drug Targets for the Treatment of Plasmodium falciparum Malaria: New Insights

“…The last two steps of the pyrimidine biosynthesis in P. falciparum are catalyzed by a heteromeric complex that consists of two homodimers of PfOPRT and PfOPDC encoded by two separate genes [174,175].…”

Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

“…It salvages the preformed purine bases/nucleosides from the human host and converts them to their mono-, di-and triphosphates. The parasite can only synthesize pyrimidines de novo from HCO 3 -, ATP, glutamine, aspartate, and 5-phosphoribosyl-1-pyrophosphate [43][44][45][46][47][48][49][50][51][52][53] . These unique properties on both purine and pyrimidine requirement of the parasite are key differences from the human host, in which both functional de novo and salvage pathways of the purine and pyrimidine synthesis exists [46,48,[54][55][56][57][58].…”

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

“…These values are similar to those obtained previously for the E. coli enzyme as well as the Mt and P. falciparum ODCases while K m is somewhat higher (sixfold) than observed for the yeast enzyme. 9,11,29,32 The D71C enzyme exhibited an 84-fold decrease in k cat and a 1071-fold decrease in catalytic efficiency compared with the wild-type enzyme (Table I). This indicates that mutants exhibiting as low as 0.1% of wild-type activity are recovered in the plasmid Figure 5.…”

“…1,2,4 Apart from its high proficiency, ODCase has also been identified as a potential drug target to develop therapeutics against RNA viruses such as West Nile Virus, 6,7 and Plasmodia parasites including the causative agent of malaria, Plasmodium falciparum. 8,9 The X-ray crystal structures of ODCase from several organisms including human, parasite, yeast, Bacillus subtilis and E. coli, among others, have been determined. 10,11 The mechanism by which ODCase achieves such a large rate enhancement over the noncatalyzed rate has been a subject of active investigation.…”