Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli.
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.
Tahar
R
R
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91198, Gif-sur-Yvette, France.
de Pécoulas
P E
PE
Basco
L K
LK
Chiadmi
M
M
Mazabraud
A
A
D000818:Animals; D003001:Cloning, Molecular; D004351:Drug Resistance; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007700:Kinetics; D010966:Plasmodium vivax / Q000201:enzymology* / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine