{"gene":"PF3D7_0512700","run_date":"2026-09-07T10:55:52","timeline":{"discoveries":[{"year":1983,"finding":"P. falciparum OPRT (orotate phosphoribosyltransferase) is a monofunctional enzyme that releases orotidylate into the medium rather than channeling it directly to orotidylate decarboxylase, and the two parasite enzymes can be resolved from each other by biospecific elution from blue Sepharose, demonstrating they do not form a bifunctional protein as in mammalian cells. P. falciparum OPRT is also much more sensitive to inhibition by mercurial reagents than the red blood cell enzyme.","method":"Blue Sepharose chromatography with biospecific elution, enzyme activity assays, inhibitor profiling with mercurial reagents","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical fractionation with two orthogonal methods (chromatographic separation and enzymatic assay), single lab","pmids":["6338005"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1980,"finding":"High activities of OPRT were demonstrated in crude supernatants of Plasmodium berghei; the enzyme is inhibited in vitro by 5-azaorotate, 5-azauracil, and 6-azauracil, with 5-azaorotate being the most effective inhibitor.","method":"Enzyme activity assays in cell-free lysates, in vitro inhibition assays","journal":"Annals of tropical medicine and parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (activity assay in crude extract), no molecular characterization","pmids":["7002072"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":1986,"finding":"Pyrazofurin (20 µM) caused 50% inhibition of P. falciparum OPRT activity in vitro, establishing that pyrazofurin acts at least in part by directly inhibiting OPRT enzymatic activity.","method":"In vitro enzyme inhibition assay with parasite cell-free extract","journal":"Molecular and biochemical parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single enzyme inhibition assay in crude extract, single lab, single method","pmids":["3515174"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"P. falciparum OPRT was cloned, expressed as a soluble recombinant protein in E. coli, and purified. The recombinant enzyme has a molecular mass of 33 kDa (monomer) and exists as a dimer (~67 kDa) by gel filtration. It contains a unique N-terminal extension of 66 amino acids and a 20-amino-acid insertion not found in other organisms. Steady-state kinetics indicate a random sequential kinetic mechanism. OPRT is the fifth enzyme of the de novo pyrimidine pathway, catalyzing formation of OMP and PPi from PRPP and orotate.","method":"Recombinant protein expression in E. coli, nickel affinity and gel-filtration chromatography, SDS-PAGE, chemical cross-linking with dimethyl suberimidate, steady-state kinetics, product inhibition studies","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — recombinant enzyme reconstitution with multiple orthogonal biochemical methods (purification, cross-linking, kinetics), single lab","pmids":["15003844"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"P. falciparum OPRT and OMPDC co-elute across all chromatographic steps during purification from P. falciparum cultured in vitro, forming a multienzyme complex of ~140 kDa consisting of two subunits each of OPRT (32 kDa) and OMPDC (38 kDa), i.e., a heterotetrameric (OPRT)2(OMPDC)2 complex. Kinetic parameters and inhibitory constants of both enzyme activities within this complex differ from those of the bifunctional human red cell UMP synthase.","method":"Multi-step chromatographic co-purification from P. falciparum lysate, native molecular weight determination, SDS-PAGE, enzyme kinetics and inhibition profiling","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-purification across multiple chromatographic steps plus native-mass and kinetic characterization, independently corroborated by a subsequent study (PMID:15683248)","pmids":["15147974"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"Recombinant PfOPRT and PfOMPDC were each expressed in E. coli and individually purified as monofunctional enzymes; chemical cross-linking with dimethyl suberimidate confirmed the two proteins associate tightly as a heterotetrameric 140-kDa (OPRT)2(OMPDC)2 complex. Kinetic parameters of this reconstituted complex matched those of the native P. falciparum enzyme but differed from those of the human bifunctional UMP synthase. The unique low-complexity insertions in each subunit are proposed to mediate complex formation.","method":"Recombinant expression in E. coli, affinity purification, chemical cross-linking (dimethyl suberimidate), SDS-PAGE, kinetic analysis, inhibition assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted complex from purified recombinant subunits, cross-linking, kinetic characterization, replicates and extends PMID:15147974","pmids":["15683248"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"The transition state of PfOPRT was determined by kinetic isotope effect (KIE) analysis using multiple isotopically labeled substrates. PfOPRT forms a late, associative DN*AN‡ transition state with complete orotate departure and a partially associative nucleophile; the C1'-O distance is ~2.1 Å at the transition state. Large [2'-3H] KIEs indicate a ribosyl 2'-C-endo conformation at the transition state. p-Nitrophenyl β-D-ribose 5'-phosphate is a nanomolar inhibitor of PfOPRT consistent with this transition-state geometry.","method":"Kinetic isotope effects (multiple isotopically labeled substrates), computational transition-state modeling, enzyme inhibition assays","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple KIE measurements with computational modeling, rigorous mechanistic analysis of catalytic mechanism in a single study","pmids":["19292447"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"The reconstituted PfOPRT-OMPDC enzyme complex expressed in E. coli exhibits 5- to 125-fold higher kinetic parameters (Km, kcat, kcat/Km) compared to the monofunctional OPRT enzyme alone. Pyrophosphate (PPi) is a potent inhibitor of the enzyme complex but has only slight inhibitory effect on the monofunctional enzyme. The complex is more thermostable than either monofunctional enzyme alone.","method":"Recombinant expression in E. coli, co-purification, kinetic parameter determination, thermal inactivation assays, inhibition assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with multiple assays, single lab","pmids":["19800871"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"FTIR spectroscopy of isotopically labeled OMP bound to PfOPRT showed that both C2=O and C4=O stretching frequencies of the orotate moiety shift downward by ~20 cm-1 upon Michaelis complex formation, indicating strong hydrogen-bond-mediated leaving group polarization. The N1 atom of PfOPRT-bound OMP shows a ΔpKa of -4.6, supporting leaving group activation via a hydrogen-bond network. PPi bound to PfOPRT exists in a trianionic state with no significant P-O bond polarization, consistent with ribocation migration toward PPi as the catalytic mechanism.","method":"Isotope-edited FTIR difference spectroscopy, homology modeling, quantum chemical calculations, enzyme complex characterization","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — isotope-edited FTIR with quantum chemical calculations provide direct mechanistic characterization of active-site interactions, single rigorous study with multiple orthogonal methods","pmids":["21067187"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"Transition-state analogues designed based on PfOPRT's highly dissociative ribocation-like transition state, including iminoribitol-pyrimidine conjugates and p-nitrophenyl riboside 5'-phosphate, bound PfOPRT with dissociation constants as low as 40–80 nM. Notably, 5'-phosphorylated analogues showed similar inhibition constants to their corresponding nucleoside derivatives, an unusual property for a nucleotide-binding enzyme. Despite nanomolar binding, these inhibitors did not kill cultured P. falciparum.","method":"In vitro enzyme inhibition assays with synthesized transition-state analogues, in silico docking, P. falciparum cell culture assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme assay with structure-based inhibitor design, single lab, negative cell-culture result explicitly reported","pmids":["24158442"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"Protein-protein interaction between PfOPRT and PfOMPDC in the heterotetrameric complex is mediated by unique low-complexity region insertions (single amino acid repeats) at the α2 and α5 helices of PfOMPDC, which are located at the OPRT-OMPDC interface. This was identified by bifunctional chemical cross-linking followed by LC-MS analysis and homology modeling.","method":"Chemical cross-linking, liquid chromatography-mass spectrometry (LC-MS), homology structural modeling","journal":"Asian Pacific journal of tropical medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-linking/MS defines interaction interface, supported by structural modeling, single lab","pmids":["24507637"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"The crystal structure of P. falciparum OPRT (PfOPRTase) was determined after removal of low-complexity sequence regions that prevented crystallization. The asymmetric unit contains four catalytic dimers (eight polypeptides total). The structure reveals unique amino acids in the PfOPRTase active sites and novel parasite-specific protein-protein interactions that occlude the catalytic active sites in asymmetric dimers, suggesting autoinhibitory regulation of PfOPRTase activity.","method":"X-ray crystallography (crystal structure determination), protein engineering (removal of low-complexity sequences)","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of PfOPRT with functional interpretation of active-site occlusion, single study but Tier 1 method","pmids":["25945715"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"P. berghei pboprt- knockout mutants show restricted growth only in the youngest reticulocytes and a severe slow-growth phenotype with reduced merozoite production, demonstrating that OPRT-dependent de novo pyrimidine biosynthesis is essential for normal blood-stage replication. The pboprt- mutants also fail to complete mosquito-stage differentiation, but retain partial virulence in mice, indicating partial pyrimidine salvage can occur in reticulocytes but not fully support development.","method":"Genetic knockout in P. berghei (pboprt-), in vivo mouse infection, mosquito transmission assay, parasitemia and developmental stage monitoring","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with multiple defined phenotypic readouts (blood-stage growth, merozoite production, mosquito stage development) across in vivo and in vitro settings","pmids":["26042734"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"finding":"A fused OMPDC-OPRT bifunctional protein was engineered and expressed in E. coli, exhibiting both enzymatic activities and functioning as a dimer. Compared to the monofunctional OPRT, kcat was enhanced up to three orders of magnitude while Km was not substantially affected, indicating that physical fusion of the two enzymes yields dramatically enhanced catalytic turnover without major change in substrate affinity.","method":"Gene fusion construction, recombinant expression in E. coli, affinity and anion-exchange chromatography purification, kinetic parameter determination","journal":"Parasitology international","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution of fused enzyme with kinetic characterization, single lab, engineered construct rather than native protein","pmids":["28389349"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"Iminoribitol bis-phosphate enantiomers (L-DIAB and D-DIAB), designed as transition-state analogues of the ribocationic form of PRPP, inhibit PfOPRT enzymatic activity in vitro.","method":"In vitro enzyme inhibition assay with synthesized iminoribitol bis-phosphate compounds","journal":"ACS chemical biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single inhibition assay for PfOPRT; structural characterization performed only on yeast APRT, not PfOPRT","pmids":["29178779"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P. falciparum OPRT (PF3D7_0512700) is the fifth enzyme of the de novo pyrimidine biosynthesis pathway, catalyzing the Mg2+-dependent condensation of PRPP and orotate to form OMP and PPi via a late, highly dissociative DN*AN‡ transition state with ribocation character and leaving-group activation through an active-site hydrogen-bond network; the enzyme is a homodimer that physically associates with OMPDC to form a heterotetrameric (OPRT)2(OMPDC)2 complex with enhanced kinetic efficiency and thermal stability, an interaction mediated by unique low-complexity insertions in OMPDC, and whose crystal structure reveals parasite-specific active-site occlusion by autologous protein-protein contacts; genetic ablation in P. berghei confirms that OPRT-dependent de novo pyrimidine synthesis is essential for normal blood-stage proliferation and mosquito-stage development."},"narrative":{"mechanistic_narrative":"PF3D7_0512700 (OPRT) is the fifth enzyme of the P. falciparum de novo pyrimidine biosynthesis pathway, catalyzing the condensation of PRPP and orotate to form OMP and PPi, and its activity is required for normal parasite proliferation [PMID:15003844, PMID:26042734]. Unlike the bifunctional mammalian UMP synthase, the parasite OPRT is a monofunctional homodimer that releases orotidylate into the medium rather than channeling it internally [PMID:6338005, PMID:15003844]. It carries a parasite-specific N-terminal extension and an internal insertion absent in orthologs and operates by a random sequential kinetic mechanism [PMID:15003844]. Rather than being a covalent bifunctional protein, OPRT physically associates with OMPDC to form a heterotetrameric (OPRT)2(OMPDC)2 complex whose kinetic and inhibition properties differ from the human enzyme; assembly is mediated by unique low-complexity insertions and confers markedly enhanced catalytic efficiency and thermostability [PMID:15147974, PMID:15683248, PMID:19800871, PMID:24507637]. Catalytically, OPRT proceeds through a late, highly dissociative ribocation-like transition state with complete orotate departure, and leaving-group activation is achieved through an active-site hydrogen-bond network that polarizes the OMP pyrimidine and drives ribocation migration toward PPi [PMID:19292447, PMID:21067187]. Its crystal structure reveals parasite-specific active-site features and autologous protein-protein contacts that occlude the catalytic sites [PMID:25945715]. Genetic ablation in P. berghei confirms that OPRT-dependent de novo pyrimidine synthesis is essential for blood-stage replication and mosquito-stage development, with only partial salvage possible in reticulocytes [PMID:26042734]. Transition-state-based inhibitors bind OPRT with nanomolar affinity in vitro but have not translated into killing of cultured parasites [PMID:24158442].","teleology":[{"year":1983,"claim":"Establishing whether the parasite pyrimidine enzymes are organized like the mammalian bifunctional protein was needed to define a distinct drug target; the parasite OPRT was shown to be monofunctional, releasing orotidylate rather than channeling it.","evidence":"Blue Sepharose biospecific elution and enzyme assays separating the two parasite activities, with mercurial inhibitor profiling","pmids":["6338005"],"confidence":"Medium","gaps":["No molecular cloning or sequence at this stage","Physical association with OMPDC not yet detected","Kinetic mechanism unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1986,"claim":"Testing whether known antimetabolites act directly on OPRT clarified the target of pyrazofurin, which inhibited parasite OPRT activity in vitro.","evidence":"In vitro enzyme inhibition assay in parasite cell-free extract (also earlier azo-pyrimidine inhibition in P. berghei lysate)","pmids":["3515174","7002072"],"confidence":"Low","gaps":["Crude-extract assays, no purified enzyme","Inhibition constants and mechanism undefined","No cellular or in vivo validation of on-target effect"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2004,"claim":"Recombinant expression settled the enzyme's oligomeric state and kinetic mechanism and revealed parasite-specific sequence features, providing a defined system for mechanistic and inhibitor work.","evidence":"Cloning and E. coli expression, gel filtration and cross-linking, steady-state kinetics and product inhibition","pmids":["15003844"],"confidence":"High","gaps":["Role of N-terminal extension and insertion unknown","No structure","Association with OMPDC not addressed in this recombinant work"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"Whether OPRT and OMPDC physically associate was answered by demonstrating co-purification from parasite lysate and reconstitution of a heterotetrameric (OPRT)2(OMPDC)2 complex from recombinant subunits, defining a non-covalent alternative to the mammalian bifunctional enzyme.","evidence":"Multi-step co-purification from P. falciparum, native mass determination, and recombinant reconstitution with dimethyl suberimidate cross-linking and kinetics","pmids":["15147974","15683248"],"confidence":"High","gaps":["Interface residues not yet mapped","Functional consequence of complex formation quantified only later","No structure of the assembled complex"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"The catalytic transition state was defined, showing a late dissociative ribocation-like geometry, which rationalized structure-based inhibitor design.","evidence":"Kinetic isotope effect measurements with computational transition-state modeling and analogue inhibition","pmids":["19292447"],"confidence":"High","gaps":["Active-site residues stabilizing the transition state not yet identified","No crystallographic confirmation"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"Quantifying the benefit of complex formation showed the assembled complex has markedly higher kinetic parameters and thermostability than monofunctional OPRT, and altered PPi sensitivity, establishing functional significance of the interaction.","evidence":"Recombinant co-expression, kinetic parameter determination, thermal inactivation and inhibition assays","pmids":["19800871"],"confidence":"Medium","gaps":["Structural basis of enhancement not resolved","In vivo relevance of complex to parasite metabolism not tested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"The mechanism of leaving-group activation was characterized, showing hydrogen-bond-mediated polarization of the OMP pyrimidine and a trianionic PPi consistent with ribocation migration.","evidence":"Isotope-edited FTIR difference spectroscopy with quantum chemical calculations on OMP and PPi bound to OPRT","pmids":["21067187"],"confidence":"High","gaps":["Specific hydrogen-bonding residues inferred from modeling, not crystallography","Relationship to complex assembly not addressed"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Whether transition-state analogues could be developed as antimalarials was tested; potent nanomolar inhibitors were obtained but failed to kill cultured parasites, exposing a target-engagement-to-efficacy gap.","evidence":"In vitro enzyme inhibition with synthesized analogues, docking, and P. falciparum culture assays","pmids":["24158442"],"confidence":"Medium","gaps":["Reason for lack of cellular killing (uptake, salvage) not determined","No confirmation of intracellular target engagement"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"The physical determinants of complex assembly were mapped, identifying low-complexity single-amino-acid-repeat insertions in OMPDC helices at the interface.","evidence":"Bifunctional chemical cross-linking with LC-MS and homology modeling","pmids":["24507637"],"confidence":"Medium","gaps":["Interface defined by cross-linking/modeling, not crystallography of the complex","Contribution of OPRT's own insertions to the interface not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"The crystal structure and a genetic knockout together established both the parasite-specific structural features of OPRT and the essentiality of OPRT-dependent de novo pyrimidine synthesis for parasite development.","evidence":"X-ray crystallography (after removal of low-complexity regions) and P. berghei pboprt- knockout with in vivo and mosquito-stage phenotyping","pmids":["25945715","26042734"],"confidence":"High","gaps":["Crystallized construct lacked low-complexity regions, so the native complex interface is not directly visualized","P. berghei knockout essentiality not formally repeated in P. falciparum","Autoinhibitory active-site occlusion not functionally validated in vivo"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"claim":"Engineering a fused OMPDC-OPRT protein and further transition-state analogues extended understanding of how proximity enhances turnover and continued inhibitor development.","evidence":"Gene fusion, recombinant expression and kinetics of the fused enzyme; in vitro inhibition by iminoribitol bis-phosphate enantiomers","pmids":["28389349","29178779"],"confidence":"Medium","gaps":["Fused construct is engineered, not native","New analogues characterized only by in vitro OPRT inhibition without cellular efficacy data"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"Why potent nanomolar OPRT transition-state inhibitors fail to kill parasites despite the genetic essentiality of the pathway remains unresolved, as does the structural basis for OPRT-OMPDC-mediated kinetic enhancement in the native complex.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the intact (OPRT)2(OMPDC)2 complex with low-complexity regions","Cellular uptake/salvage limits on inhibitor efficacy undetermined","Physiological role of proposed autoinhibitory active-site occlusion untested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Heterotetrameric (OPRT)2(OMPDC)2 orotate phosphoribosyltransferase-orotidine-5'-phosphate decarboxylase complex"],"partners":["OMPDC (OROTIDINE-5'-PHOSPHATE DECARBOXYLASE, P. FALCIPARUM)"],"other_free_text":["Monofunctional homodimer that releases orotidylate rather than channeling to OMPDC, unlike the bifunctional mammalian UMP synthase [#0, #3]","Contains a parasite-specific N-terminal extension (~66 aa) and a 20-aa insertion; complex formation attributed to unique low-complexity insertions [#3, #5, #10]","Random sequential kinetic mechanism [#3]","Late, highly dissociative ribocation-like DN*AN transition state with hydrogen-bond-mediated leaving-group activation and PPi as ribocation acceptor [#6, #8]","Complex formation enhances kcat/Km and thermostability and alters PPi inhibition relative to monofunctional OPRT [#7]","Crystal structure shows parasite-specific active sites and autologous protein-protein contacts occluding catalytic sites, suggesting autoinhibitory regulation [#11]","Genetically essential for P. berghei blood-stage replication and mosquito-stage development [#12]","Inhibited in vitro by pyrazofurin, azo-pyrimidines, and nanomolar transition-state analogues, though analogues do not kill cultured P. falciparum [#1, #2, #9, #14]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"26042734","id":"PMC_26042734","title":"Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites.","date":"2015","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26042734","citation_count":62,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19292447","id":"PMC_19292447","title":"Transition states of Plasmodium falciparum and human orotate phosphoribosyltransferases.","date":"2009","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/19292447","citation_count":42,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15683248","id":"PMC_15683248","title":"A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15683248","citation_count":42,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"3515174","id":"PMC_3515174","title":"In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo.","date":"1986","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/3515174","citation_count":42,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15003844","id":"PMC_15003844","title":"Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile.","date":"2004","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15003844","citation_count":39,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"6338005","id":"PMC_6338005","title":"Orotidylate-metabolizing enzymes of the human malarial parasite, Plasmodium falciparum, differ from host cell enzymes.","date":"1983","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/6338005","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15147974","id":"PMC_15147974","title":"Orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15147974","citation_count":26,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27262062","id":"PMC_27262062","title":"Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.","date":"2016","source":"Asian Pacific journal of tropical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27262062","citation_count":24,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19230569","id":"PMC_19230569","title":"Molecular biology and biochemistry of malarial parasite pyrimidine biosynthetic pathway.","date":"2003","source":"The Southeast Asian journal of tropical medicine and public health","url":"https://pubmed.ncbi.nlm.nih.gov/19230569","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"6277049","id":"PMC_6277049","title":"Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase in Plasmodium falciparum.","date":"1981","source":"Transactions of the Royal Society of Tropical Medicine and Hygiene","url":"https://pubmed.ncbi.nlm.nih.gov/6277049","citation_count":17,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24158442","id":"PMC_24158442","title":"Transition state analogues of Plasmodium falciparum and human orotate phosphoribosyltransferases.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24158442","citation_count":14,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"7002072","id":"PMC_7002072","title":"Biosynthesis of uridine monophosphate in Plasmodium berghei.","date":"1980","source":"Annals of tropical medicine and parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/7002072","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21067187","id":"PMC_21067187","title":"Leaving group activation and pyrophosphate ionic state at the catalytic site of Plasmodium falciparum orotate phosphoribosyltransferase.","date":"2010","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/21067187","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19800871","id":"PMC_19800871","title":"Kinetic benefits and thermal stability of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase enzyme complex in human malaria parasite Plasmodium falciparum.","date":"2009","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19800871","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24507637","id":"PMC_24507637","title":"A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase.","date":"2014","source":"Asian Pacific journal of tropical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/24507637","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15571277","id":"PMC_15571277","title":"Cloning and expression of malarial pyrimidine enzymes.","date":"2004","source":"Nucleosides, nucleotides & nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/15571277","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25945715","id":"PMC_25945715","title":"Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein-protein interactions.","date":"2015","source":"Acta crystallographica. Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/25945715","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22298010","id":"PMC_22298010","title":"Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum.","date":"2012","source":"Acta crystallographica. Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/22298010","citation_count":5,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29178779","id":"PMC_29178779","title":"Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase.","date":"2017","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/29178779","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"28389349","id":"PMC_28389349","title":"Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase.","date":"2017","source":"Parasitology international","url":"https://pubmed.ncbi.nlm.nih.gov/28389349","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["OPRT","orotate phosphoribosyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12773,"output_tokens":4531,"usd":0.053142,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11003,"output_tokens":4693,"usd":0.08617,"stage2_stop_reason":"end_turn"},"total_usd":0.139312,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1983,\n      \"finding\": \"P. falciparum OPRT (orotate phosphoribosyltransferase) is a monofunctional enzyme that releases orotidylate into the medium rather than channeling it directly to orotidylate decarboxylase, and the two parasite enzymes can be resolved from each other by biospecific elution from blue Sepharose, demonstrating they do not form a bifunctional protein as in mammalian cells. P. falciparum OPRT is also much more sensitive to inhibition by mercurial reagents than the red blood cell enzyme.\",\n      \"method\": \"Blue Sepharose chromatography with biospecific elution, enzyme activity assays, inhibitor profiling with mercurial reagents\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical fractionation with two orthogonal methods (chromatographic separation and enzymatic assay), single lab\",\n      \"pmids\": [\"6338005\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1980,\n      \"finding\": \"High activities of OPRT were demonstrated in crude supernatants of Plasmodium berghei; the enzyme is inhibited in vitro by 5-azaorotate, 5-azauracil, and 6-azauracil, with 5-azaorotate being the most effective inhibitor.\",\n      \"method\": \"Enzyme activity assays in cell-free lysates, in vitro inhibition assays\",\n      \"journal\": \"Annals of tropical medicine and parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (activity assay in crude extract), no molecular characterization\",\n      \"pmids\": [\"7002072\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Pyrazofurin (20 µM) caused 50% inhibition of P. falciparum OPRT activity in vitro, establishing that pyrazofurin acts at least in part by directly inhibiting OPRT enzymatic activity.\",\n      \"method\": \"In vitro enzyme inhibition assay with parasite cell-free extract\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single enzyme inhibition assay in crude extract, single lab, single method\",\n      \"pmids\": [\"3515174\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"P. falciparum OPRT was cloned, expressed as a soluble recombinant protein in E. coli, and purified. The recombinant enzyme has a molecular mass of 33 kDa (monomer) and exists as a dimer (~67 kDa) by gel filtration. It contains a unique N-terminal extension of 66 amino acids and a 20-amino-acid insertion not found in other organisms. Steady-state kinetics indicate a random sequential kinetic mechanism. OPRT is the fifth enzyme of the de novo pyrimidine pathway, catalyzing formation of OMP and PPi from PRPP and orotate.\",\n      \"method\": \"Recombinant protein expression in E. coli, nickel affinity and gel-filtration chromatography, SDS-PAGE, chemical cross-linking with dimethyl suberimidate, steady-state kinetics, product inhibition studies\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — recombinant enzyme reconstitution with multiple orthogonal biochemical methods (purification, cross-linking, kinetics), single lab\",\n      \"pmids\": [\"15003844\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"P. falciparum OPRT and OMPDC co-elute across all chromatographic steps during purification from P. falciparum cultured in vitro, forming a multienzyme complex of ~140 kDa consisting of two subunits each of OPRT (32 kDa) and OMPDC (38 kDa), i.e., a heterotetrameric (OPRT)2(OMPDC)2 complex. Kinetic parameters and inhibitory constants of both enzyme activities within this complex differ from those of the bifunctional human red cell UMP synthase.\",\n      \"method\": \"Multi-step chromatographic co-purification from P. falciparum lysate, native molecular weight determination, SDS-PAGE, enzyme kinetics and inhibition profiling\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-purification across multiple chromatographic steps plus native-mass and kinetic characterization, independently corroborated by a subsequent study (PMID:15683248)\",\n      \"pmids\": [\"15147974\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Recombinant PfOPRT and PfOMPDC were each expressed in E. coli and individually purified as monofunctional enzymes; chemical cross-linking with dimethyl suberimidate confirmed the two proteins associate tightly as a heterotetrameric 140-kDa (OPRT)2(OMPDC)2 complex. Kinetic parameters of this reconstituted complex matched those of the native P. falciparum enzyme but differed from those of the human bifunctional UMP synthase. The unique low-complexity insertions in each subunit are proposed to mediate complex formation.\",\n      \"method\": \"Recombinant expression in E. coli, affinity purification, chemical cross-linking (dimethyl suberimidate), SDS-PAGE, kinetic analysis, inhibition assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted complex from purified recombinant subunits, cross-linking, kinetic characterization, replicates and extends PMID:15147974\",\n      \"pmids\": [\"15683248\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The transition state of PfOPRT was determined by kinetic isotope effect (KIE) analysis using multiple isotopically labeled substrates. PfOPRT forms a late, associative DN*AN‡ transition state with complete orotate departure and a partially associative nucleophile; the C1'-O distance is ~2.1 Å at the transition state. Large [2'-3H] KIEs indicate a ribosyl 2'-C-endo conformation at the transition state. p-Nitrophenyl β-D-ribose 5'-phosphate is a nanomolar inhibitor of PfOPRT consistent with this transition-state geometry.\",\n      \"method\": \"Kinetic isotope effects (multiple isotopically labeled substrates), computational transition-state modeling, enzyme inhibition assays\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple KIE measurements with computational modeling, rigorous mechanistic analysis of catalytic mechanism in a single study\",\n      \"pmids\": [\"19292447\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The reconstituted PfOPRT-OMPDC enzyme complex expressed in E. coli exhibits 5- to 125-fold higher kinetic parameters (Km, kcat, kcat/Km) compared to the monofunctional OPRT enzyme alone. Pyrophosphate (PPi) is a potent inhibitor of the enzyme complex but has only slight inhibitory effect on the monofunctional enzyme. The complex is more thermostable than either monofunctional enzyme alone.\",\n      \"method\": \"Recombinant expression in E. coli, co-purification, kinetic parameter determination, thermal inactivation assays, inhibition assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with multiple assays, single lab\",\n      \"pmids\": [\"19800871\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"FTIR spectroscopy of isotopically labeled OMP bound to PfOPRT showed that both C2=O and C4=O stretching frequencies of the orotate moiety shift downward by ~20 cm-1 upon Michaelis complex formation, indicating strong hydrogen-bond-mediated leaving group polarization. The N1 atom of PfOPRT-bound OMP shows a ΔpKa of -4.6, supporting leaving group activation via a hydrogen-bond network. PPi bound to PfOPRT exists in a trianionic state with no significant P-O bond polarization, consistent with ribocation migration toward PPi as the catalytic mechanism.\",\n      \"method\": \"Isotope-edited FTIR difference spectroscopy, homology modeling, quantum chemical calculations, enzyme complex characterization\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — isotope-edited FTIR with quantum chemical calculations provide direct mechanistic characterization of active-site interactions, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"21067187\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Transition-state analogues designed based on PfOPRT's highly dissociative ribocation-like transition state, including iminoribitol-pyrimidine conjugates and p-nitrophenyl riboside 5'-phosphate, bound PfOPRT with dissociation constants as low as 40–80 nM. Notably, 5'-phosphorylated analogues showed similar inhibition constants to their corresponding nucleoside derivatives, an unusual property for a nucleotide-binding enzyme. Despite nanomolar binding, these inhibitors did not kill cultured P. falciparum.\",\n      \"method\": \"In vitro enzyme inhibition assays with synthesized transition-state analogues, in silico docking, P. falciparum cell culture assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme assay with structure-based inhibitor design, single lab, negative cell-culture result explicitly reported\",\n      \"pmids\": [\"24158442\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Protein-protein interaction between PfOPRT and PfOMPDC in the heterotetrameric complex is mediated by unique low-complexity region insertions (single amino acid repeats) at the α2 and α5 helices of PfOMPDC, which are located at the OPRT-OMPDC interface. This was identified by bifunctional chemical cross-linking followed by LC-MS analysis and homology modeling.\",\n      \"method\": \"Chemical cross-linking, liquid chromatography-mass spectrometry (LC-MS), homology structural modeling\",\n      \"journal\": \"Asian Pacific journal of tropical medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-linking/MS defines interaction interface, supported by structural modeling, single lab\",\n      \"pmids\": [\"24507637\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The crystal structure of P. falciparum OPRT (PfOPRTase) was determined after removal of low-complexity sequence regions that prevented crystallization. The asymmetric unit contains four catalytic dimers (eight polypeptides total). The structure reveals unique amino acids in the PfOPRTase active sites and novel parasite-specific protein-protein interactions that occlude the catalytic active sites in asymmetric dimers, suggesting autoinhibitory regulation of PfOPRTase activity.\",\n      \"method\": \"X-ray crystallography (crystal structure determination), protein engineering (removal of low-complexity sequences)\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of PfOPRT with functional interpretation of active-site occlusion, single study but Tier 1 method\",\n      \"pmids\": [\"25945715\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"P. berghei pboprt- knockout mutants show restricted growth only in the youngest reticulocytes and a severe slow-growth phenotype with reduced merozoite production, demonstrating that OPRT-dependent de novo pyrimidine biosynthesis is essential for normal blood-stage replication. The pboprt- mutants also fail to complete mosquito-stage differentiation, but retain partial virulence in mice, indicating partial pyrimidine salvage can occur in reticulocytes but not fully support development.\",\n      \"method\": \"Genetic knockout in P. berghei (pboprt-), in vivo mouse infection, mosquito transmission assay, parasitemia and developmental stage monitoring\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with multiple defined phenotypic readouts (blood-stage growth, merozoite production, mosquito stage development) across in vivo and in vitro settings\",\n      \"pmids\": [\"26042734\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A fused OMPDC-OPRT bifunctional protein was engineered and expressed in E. coli, exhibiting both enzymatic activities and functioning as a dimer. Compared to the monofunctional OPRT, kcat was enhanced up to three orders of magnitude while Km was not substantially affected, indicating that physical fusion of the two enzymes yields dramatically enhanced catalytic turnover without major change in substrate affinity.\",\n      \"method\": \"Gene fusion construction, recombinant expression in E. coli, affinity and anion-exchange chromatography purification, kinetic parameter determination\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution of fused enzyme with kinetic characterization, single lab, engineered construct rather than native protein\",\n      \"pmids\": [\"28389349\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Iminoribitol bis-phosphate enantiomers (L-DIAB and D-DIAB), designed as transition-state analogues of the ribocationic form of PRPP, inhibit PfOPRT enzymatic activity in vitro.\",\n      \"method\": \"In vitro enzyme inhibition assay with synthesized iminoribitol bis-phosphate compounds\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single inhibition assay for PfOPRT; structural characterization performed only on yeast APRT, not PfOPRT\",\n      \"pmids\": [\"29178779\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"P. falciparum OPRT (PF3D7_0512700) is the fifth enzyme of the de novo pyrimidine biosynthesis pathway, catalyzing the Mg2+-dependent condensation of PRPP and orotate to form OMP and PPi via a late, highly dissociative DN*AN‡ transition state with ribocation character and leaving-group activation through an active-site hydrogen-bond network; the enzyme is a homodimer that physically associates with OMPDC to form a heterotetrameric (OPRT)2(OMPDC)2 complex with enhanced kinetic efficiency and thermal stability, an interaction mediated by unique low-complexity insertions in OMPDC, and whose crystal structure reveals parasite-specific active-site occlusion by autologous protein-protein contacts; genetic ablation in P. berghei confirms that OPRT-dependent de novo pyrimidine synthesis is essential for normal blood-stage proliferation and mosquito-stage development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0512700 (OPRT) is the fifth enzyme of the P. falciparum de novo pyrimidine biosynthesis pathway, catalyzing the condensation of PRPP and orotate to form OMP and PPi, and its activity is required for normal parasite proliferation [#3, #12]. Unlike the bifunctional mammalian UMP synthase, the parasite OPRT is a monofunctional homodimer that releases orotidylate into the medium rather than channeling it internally [#0, #3]. It carries a parasite-specific N-terminal extension and an internal insertion absent in orthologs and operates by a random sequential kinetic mechanism [#3]. Rather than being a covalent bifunctional protein, OPRT physically associates with OMPDC to form a heterotetrameric (OPRT)2(OMPDC)2 complex whose kinetic and inhibition properties differ from the human enzyme; assembly is mediated by unique low-complexity insertions and confers markedly enhanced catalytic efficiency and thermostability [#4, #5, #7, #10]. Catalytically, OPRT proceeds through a late, highly dissociative ribocation-like transition state with complete orotate departure, and leaving-group activation is achieved through an active-site hydrogen-bond network that polarizes the OMP pyrimidine and drives ribocation migration toward PPi [#6, #8]. Its crystal structure reveals parasite-specific active-site features and autologous protein-protein contacts that occlude the catalytic sites [#11]. Genetic ablation in P. berghei confirms that OPRT-dependent de novo pyrimidine synthesis is essential for blood-stage replication and mosquito-stage development, with only partial salvage possible in reticulocytes [#12]. Transition-state-based inhibitors bind OPRT with nanomolar affinity in vitro but have not translated into killing of cultured parasites [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1983,\n      \"claim\": \"Establishing whether the parasite pyrimidine enzymes are organized like the mammalian bifunctional protein was needed to define a distinct drug target; the parasite OPRT was shown to be monofunctional, releasing orotidylate rather than channeling it.\",\n      \"evidence\": \"Blue Sepharose biospecific elution and enzyme assays separating the two parasite activities, with mercurial inhibitor profiling\",\n      \"pmids\": [\"6338005\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No molecular cloning or sequence at this stage\", \"Physical association with OMPDC not yet detected\", \"Kinetic mechanism unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Testing whether known antimetabolites act directly on OPRT clarified the target of pyrazofurin, which inhibited parasite OPRT activity in vitro.\",\n      \"evidence\": \"In vitro enzyme inhibition assay in parasite cell-free extract (also earlier azo-pyrimidine inhibition in P. berghei lysate)\",\n      \"pmids\": [\"3515174\", \"7002072\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Crude-extract assays, no purified enzyme\", \"Inhibition constants and mechanism undefined\", \"No cellular or in vivo validation of on-target effect\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Recombinant expression settled the enzyme's oligomeric state and kinetic mechanism and revealed parasite-specific sequence features, providing a defined system for mechanistic and inhibitor work.\",\n      \"evidence\": \"Cloning and E. coli expression, gel filtration and cross-linking, steady-state kinetics and product inhibition\",\n      \"pmids\": [\"15003844\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Role of N-terminal extension and insertion unknown\", \"No structure\", \"Association with OMPDC not addressed in this recombinant work\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Whether OPRT and OMPDC physically associate was answered by demonstrating co-purification from parasite lysate and reconstitution of a heterotetrameric (OPRT)2(OMPDC)2 complex from recombinant subunits, defining a non-covalent alternative to the mammalian bifunctional enzyme.\",\n      \"evidence\": \"Multi-step co-purification from P. falciparum, native mass determination, and recombinant reconstitution with dimethyl suberimidate cross-linking and kinetics\",\n      \"pmids\": [\"15147974\", \"15683248\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Interface residues not yet mapped\", \"Functional consequence of complex formation quantified only later\", \"No structure of the assembled complex\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The catalytic transition state was defined, showing a late dissociative ribocation-like geometry, which rationalized structure-based inhibitor design.\",\n      \"evidence\": \"Kinetic isotope effect measurements with computational transition-state modeling and analogue inhibition\",\n      \"pmids\": [\"19292447\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Active-site residues stabilizing the transition state not yet identified\", \"No crystallographic confirmation\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Quantifying the benefit of complex formation showed the assembled complex has markedly higher kinetic parameters and thermostability than monofunctional OPRT, and altered PPi sensitivity, establishing functional significance of the interaction.\",\n      \"evidence\": \"Recombinant co-expression, kinetic parameter determination, thermal inactivation and inhibition assays\",\n      \"pmids\": [\"19800871\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of enhancement not resolved\", \"In vivo relevance of complex to parasite metabolism not tested\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The mechanism of leaving-group activation was characterized, showing hydrogen-bond-mediated polarization of the OMP pyrimidine and a trianionic PPi consistent with ribocation migration.\",\n      \"evidence\": \"Isotope-edited FTIR difference spectroscopy with quantum chemical calculations on OMP and PPi bound to OPRT\",\n      \"pmids\": [\"21067187\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Specific hydrogen-bonding residues inferred from modeling, not crystallography\", \"Relationship to complex assembly not addressed\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Whether transition-state analogues could be developed as antimalarials was tested; potent nanomolar inhibitors were obtained but failed to kill cultured parasites, exposing a target-engagement-to-efficacy gap.\",\n      \"evidence\": \"In vitro enzyme inhibition with synthesized analogues, docking, and P. falciparum culture assays\",\n      \"pmids\": [\"24158442\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Reason for lack of cellular killing (uptake, salvage) not determined\", \"No confirmation of intracellular target engagement\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The physical determinants of complex assembly were mapped, identifying low-complexity single-amino-acid-repeat insertions in OMPDC helices at the interface.\",\n      \"evidence\": \"Bifunctional chemical cross-linking with LC-MS and homology modeling\",\n      \"pmids\": [\"24507637\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Interface defined by cross-linking/modeling, not crystallography of the complex\", \"Contribution of OPRT's own insertions to the interface not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The crystal structure and a genetic knockout together established both the parasite-specific structural features of OPRT and the essentiality of OPRT-dependent de novo pyrimidine synthesis for parasite development.\",\n      \"evidence\": \"X-ray crystallography (after removal of low-complexity regions) and P. berghei pboprt- knockout with in vivo and mosquito-stage phenotyping\",\n      \"pmids\": [\"25945715\", \"26042734\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Crystallized construct lacked low-complexity regions, so the native complex interface is not directly visualized\", \"P. berghei knockout essentiality not formally repeated in P. falciparum\", \"Autoinhibitory active-site occlusion not functionally validated in vivo\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Engineering a fused OMPDC-OPRT protein and further transition-state analogues extended understanding of how proximity enhances turnover and continued inhibitor development.\",\n      \"evidence\": \"Gene fusion, recombinant expression and kinetics of the fused enzyme; in vitro inhibition by iminoribitol bis-phosphate enantiomers\",\n      \"pmids\": [\"28389349\", \"29178779\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Fused construct is engineered, not native\", \"New analogues characterized only by in vitro OPRT inhibition without cellular efficacy data\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Why potent nanomolar OPRT transition-state inhibitors fail to kill parasites despite the genetic essentiality of the pathway remains unresolved, as does the structural basis for OPRT-OMPDC-mediated kinetic enhancement in the native complex.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structure of the intact (OPRT)2(OMPDC)2 complex with low-complexity regions\", \"Cellular uptake/salvage limits on inhibitor efficacy undetermined\", \"Physiological role of proposed autoinhibitory active-site occlusion untested\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004588\", \"supporting_discovery_ids\": [0, 3, 4, 5, 6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044205\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"complexes\": [\n      \"Heterotetrameric (OPRT)2(OMPDC)2 orotate phosphoribosyltransferase-orotidine-5'-phosphate decarboxylase complex\"\n    ],\n    \"partners\": [\n      \"OMPDC (orotidine-5'-phosphate decarboxylase, P. falciparum)\"\n    ],\n    \"other_free_text\": [\n      \"Monofunctional homodimer that releases orotidylate rather than channeling to OMPDC, unlike the bifunctional mammalian UMP synthase [#0, #3]\",\n      \"Contains a parasite-specific N-terminal extension (~66 aa) and a 20-aa insertion; complex formation attributed to unique low-complexity insertions [#3, #5, #10]\",\n      \"Random sequential kinetic mechanism [#3]\",\n      \"Late, highly dissociative ribocation-like DN*AN transition state with hydrogen-bond-mediated leaving-group activation and PPi as ribocation acceptor [#6, #8]\",\n      \"Complex formation enhances kcat/Km and thermostability and alters PPi inhibition relative to monofunctional OPRT [#7]\",\n      \"Crystal structure shows parasite-specific active sites and autologous protein-protein contacts occluding catalytic sites, suggesting autoinhibitory regulation [#11]\",\n      \"Genetically essential for P. berghei blood-stage replication and mosquito-stage development [#12]\",\n      \"Inhibited in vitro by pyrazofurin, azo-pyrimidines, and nanomolar transition-state analogues, though analogues do not kill cultured P. falciparum [#1, #2, #9, #14]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0512700","name":null,"product":"orotate phosphoribosyltransferase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1112400","name":null,"product":"orotate phosphoribosyltransferase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1020400","name":null,"product":"orotate phosphoribosyltransferase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"OPRT","evaluation":null}