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

Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). in Plasmo dium falciparum antifolate resistance has been associated with point mutati ons in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagate d in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escher ichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. roil. The soluble, active DHFR recovered after d enaturation and maturation was purified to homogeneity by affinity chromato graphy. Kinetic properties oi 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 prog uanil (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 r esistance. (C) 2001 Elsevier Science B.V. All rights reserved.