title: Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.
abstract: Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.
authors.affiliation: Department of Chemistry, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Srisuda
authors.initials: S
authors.lastname: Tirakarn
authors.forename: Pinpunya
authors.initials: P
authors.lastname: Riangrungroj
authors.forename: Palangpon
authors.initials: P
authors.lastname: Kongsaeree
authors.forename: Mallika
authors.initials: M
authors.lastname: Imwong
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.forename: Ubolsree
authors.initials: U
authors.lastname: Leartsakulpanich
mesh_terms: D000595:Amino Acid Sequence; D000962:Antimalarials / Q000494:pharmacology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D041122:Plasmodium ovale / Q000201:enzymology* / Q000235:genetics; D002727:Proguanil / Q000494:pharmacology; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011739:Pyrimethamine / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014227:Triazines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; D015800:Protozoan Proteins; D014227:Triazines; C056930:thymidylate synthase-dihydrofolate reductase; C026009:cycloguanil; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D002727:Proguanil; D011739:Pyrimethamine