| 1983 |
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. |
Blue Sepharose chromatography with biospecific elution, enzyme activity assays, inhibitor profiling with mercurial reagents |
The Journal of biological chemistry |
Medium |
6338005
|
| 1980 |
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. |
Enzyme activity assays in cell-free lysates, in vitro inhibition assays |
Annals of tropical medicine and parasitology |
Low |
7002072
|
| 1986 |
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. |
In vitro enzyme inhibition assay with parasite cell-free extract |
Molecular and biochemical parasitology |
Low |
3515174
|
| 2004 |
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. |
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 |
Molecular and biochemical parasitology |
High |
15003844
|
| 2004 |
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. |
Multi-step chromatographic co-purification from P. falciparum lysate, native molecular weight determination, SDS-PAGE, enzyme kinetics and inhibition profiling |
Biochemical and biophysical research communications |
High |
15147974
|
| 2005 |
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. |
Recombinant expression in E. coli, affinity purification, chemical cross-linking (dimethyl suberimidate), SDS-PAGE, kinetic analysis, inhibition assays |
Biochemistry |
High |
15683248
|
| 2009 |
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. |
Kinetic isotope effects (multiple isotopically labeled substrates), computational transition-state modeling, enzyme inhibition assays |
Journal of the American Chemical Society |
High |
19292447
|
| 2009 |
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. |
Recombinant expression in E. coli, co-purification, kinetic parameter determination, thermal inactivation assays, inhibition assays |
Biochemical and biophysical research communications |
Medium |
19800871
|
| 2010 |
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. |
Isotope-edited FTIR difference spectroscopy, homology modeling, quantum chemical calculations, enzyme complex characterization |
Journal of the American Chemical Society |
High |
21067187
|
| 2013 |
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. |
In vitro enzyme inhibition assays with synthesized transition-state analogues, in silico docking, P. falciparum cell culture assay |
The Journal of biological chemistry |
Medium |
24158442
|
| 2014 |
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. |
Chemical cross-linking, liquid chromatography-mass spectrometry (LC-MS), homology structural modeling |
Asian Pacific journal of tropical medicine |
Medium |
24507637
|
| 2015 |
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. |
X-ray crystallography (crystal structure determination), protein engineering (removal of low-complexity sequences) |
Acta crystallographica. Section F, Structural biology communications |
High |
25945715
|
| 2015 |
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. |
Genetic knockout in P. berghei (pboprt-), in vivo mouse infection, mosquito transmission assay, parasitemia and developmental stage monitoring |
PLoS pathogens |
High |
26042734
|
| 2017 |
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. |
Gene fusion construction, recombinant expression in E. coli, affinity and anion-exchange chromatography purification, kinetic parameter determination |
Parasitology international |
Medium |
28389349
|
| 2017 |
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. |
In vitro enzyme inhibition assay with synthesized iminoribitol bis-phosphate compounds |
ACS chemical biology |
Low |
29178779
|