Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli. 2001

R Tahar, and P E de Pécoulas, and L K Basco, and M Chiadmi, and A Mazabraud
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91198, Gif-sur-Yvette, France.

Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. coli. The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography. Kinetic properties of the recombinant P. vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH. Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected. The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D010966 Plasmodium vivax A protozoan parasite that causes vivax malaria (MALARIA, VIVAX). This species is found almost everywhere malaria is endemic and is the only one that has a range extending into the temperate regions. Plasmodium vivaxs,vivax, Plasmodium
D011739 Pyrimethamine One of the FOLIC ACID ANTAGONISTS that is used as an antimalarial or with a sulfonamide to treat toxoplasmosis. Chloridin,Daraprim,Malocide,Tindurine
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004351 Drug Resistance Diminished or failed response of an organism, disease or tissue to the intended effectiveness of a chemical or drug. It should be differentiated from DRUG TOLERANCE which is the progressive diminution of the susceptibility of a human or animal to the effects of a drug, as a result of continued administration. Resistance, Drug
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005493 Folic Acid Antagonists Inhibitors of the enzyme, dihydrofolate reductase (TETRAHYDROFOLATE DEHYDROGENASE), which converts dihydrofolate (FH2) to tetrahydrofolate (FH4). They are frequently used in cancer chemotherapy. (From AMA, Drug Evaluations Annual, 1994, p2033) Antifolate,Antifolates,Dihydrofolate Reductase Inhibitor,Folic Acid Antagonist,Dihydrofolate Reductase Inhibitors,Folic Acid Metabolism Inhibitors,Acid Antagonist, Folic,Acid Antagonists, Folic,Antagonist, Folic Acid,Antagonists, Folic Acid,Inhibitor, Dihydrofolate Reductase,Inhibitors, Dihydrofolate Reductase,Reductase Inhibitor, Dihydrofolate,Reductase Inhibitors, Dihydrofolate
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013762 Tetrahydrofolate Dehydrogenase An enzyme of the oxidoreductase class that catalyzes the reaction 7,8-dihyrofolate and NADPH to yield 5,6,7,8-tetrahydrofolate and NADPH+, producing reduced folate for amino acid metabolism, purine ring synthesis, and the formation of deoxythymidine monophosphate. Methotrexate and other folic acid antagonists used as chemotherapeutic drugs act by inhibiting this enzyme. (Dorland, 27th ed) EC 1.5.1.3. Dihydrofolate Dehydrogenase,Dihydrofolate Reductase,Folic Acid Reductase,Acid Reductase, Folic,Dehydrogenase, Dihydrofolate,Dehydrogenase, Tetrahydrofolate,Reductase, Dihydrofolate,Reductase, Folic Acid

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