Manee ChanamaPenchit ChitnumsubYongyuth YuthavongMahidol UniversityThailand National Center for Genetic Engineering and Biotechnology2018-06-212018-06-212005-01-01Molecular and Biochemical Parasitology. Vol.139, No.1 (2005), 83-90016668512-s2.0-11144309676https://repository.li.mahidol.ac.th/handle/20.500.14594/16392Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph χ2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS kcatvalue of the R470D + C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Kmvalues for dUMP and CH2H4folate of the R470D + C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D + C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS. © 2004 Elsevier B.V. All rights reserved.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyArticleSCOPUS10.1016/j.molbiopara.2004.09.010