ABSTRACT
Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.
Subject(s)
Enzyme Inhibitors/chemistry , Orotidine-5'-Phosphate Decarboxylase/antagonists & inhibitors , Orotidine-5'-Phosphate Decarboxylase/chemistry , Plasmodium falciparum/enzymology , Antimalarials/chemistry , Antimalarials/pharmacology , Binding Sites , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Models, Molecular , Orotidine-5'-Phosphate Decarboxylase/metabolism , Phenylbutyrates/chemistry , Phenylbutyrates/pharmacology , Protein Conformation , Pyrimidines/chemistry , Structure-Activity Relationship , Uridine Monophosphate/chemistryABSTRACT
Orotate phosphoribosyltransferase (OPRT) catalyzes the Mg(2+)-dependent condensation of orotic acid (OA) with 5-α-D-phosphorylribose 1-diphosphate (PRPP) to yield diphosphate (PP(i)) and the nucleotide orotidine 5'-monophosphate. OPRT from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg(2+). The crystal exhibited tetragonal symmetry, belonging to space group P4(1) or P4(3), with unit-cell parameters a = b = 49.15, c = 226.94 Å. X-ray diffraction data were collected to 2.5 Å resolution at 100 K using a synchrotron-radiation source.
Subject(s)
Orotate Phosphoribosyltransferase/chemistry , Plasmodium falciparum/enzymology , Crystallization , Crystallography, X-RayABSTRACT
Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H(2) (PGH(2)), which is a common intermediate of various prostanoids, to form PGF(2α). AKR1B3 also reduces PGH(2) to PGD(2) in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/chemistry , Animals , Crystallization , Crystallography, X-Ray , MiceABSTRACT
When 2'-deoxyadenosine was treated with HNO(2) or NO, a small amount of a previously unidentified product was formed. The product was also formed by the reaction of 2'-deoxyadenosine with isoamyl nitrite in tetrahydrofuran as a major product. The product was identified as a diazoate derivative of 2'-deoxyadenosine, a reaction intermediate. At the initial stage of the HNO(2) or NO reaction, the concentration of the diazoate was greater than or comparable to 2'-deoxyinosine, a deamination product of 2'-deoxyadenosine. The diazoate was fairly stable and decomposed with a half-life of 66h at pH 7.4 and 37 degrees C. These results suggest that the diazoate can be formed in cellular nucleosides or DNA with biologically relevant dose of HNO(2) and NO.
