Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax.
The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli. The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined. Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme. The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were approximately 60 to >4000 times less active against the mutant enzymes. In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred approximately 4000- and approximately 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR. The S58R+S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Km value than the single mutant. The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants. Data on kinetic parameters and inhibitory constant suggest that the wild-type P. vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P. vivax are responsible for antifolate resistance.
Leartsakulpanich
Ubolsree
U
National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Rama 6 Road, 10400, Bangkok, Thailand.
Imwong
Mallika
M
Pukrittayakamee
Sasithon
S
White
Nicholas J
NJ
Snounou
Georges
G
Sirawaraporn
Worachart
W
Yuthavong
Yongyuth
Y
D000818:Animals; D002384:Catalysis / Q000187:drug effects; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D010966:Plasmodium vivax / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D005493:Folic Acid Antagonists; D012965:Sodium Chloride; D011189:Potassium Chloride; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase