title: Formation of catalytically active cross-species heterodimers of thymidylate synthase from Plasmodium falciparum and Plasmodium vivax.
abstract: Thymidylate synthase (TS) of Plasmodium dihydrofolate reductase-thymidylate synthase (DHFR-TS) functions as a homodimeric enzyme with two active sites located near the subunit interface. The dimerization is essential for catalysis, since the active site of each subunit contains amino acid residues contributed from the other TS domain. In P. falciparum DHFR-TS, it has been shown that the active sites require Cys-490 from one domain and Arg-470 donated from the other domain. Mutants of these two series can complement one another giving rise to active enzyme. Here, the potential to form cross-species heterodimers between P. falciparum and P. vivax TS has been explored. Formation of cross-species heterodimer was tested by co-transformation of TS-inactive Cys-490 mutants of P. falciparum or P. vivax with corresponding TS-inactive Arg-486 mutants of P. vivax or P. falciparum into thymidine-requiring Escherichia coli. Active heterodimers were detected by subunit complementation and 6-[(3)H]-FdUMP binding assays. All combinations of the mutants tested, except for (Pf)R470A+(Pv)C506Y, were able to form catalytically active cross-species heterodimers. The single active site formed by (Pf)R470D+(Pv)C506Y and (Pv)R486D+(Pf)C490A pairs of cross-species heterodimers has k(cat) and K(m) values similar to those of intra-species heterodimers of P. falciparum and P. vivax. This is the first report to demonstrate that the TS subunit interface between Plasmodium species is sufficiently conserved to allow formation of fully active cross-species heterodimer.
authors.affiliation: Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, 10400, Thailand. phmcn@mahidol.ac.th
authors.forename: Manee
authors.initials: M
authors.lastname: Chanama
authors.forename: Suchart
authors.initials: S
authors.lastname: Chanama
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Shaw
authors.forename: Penchit
authors.initials: P
authors.lastname: Chitnumsub
authors.forename: Ubolsree
authors.initials: U
authors.lastname: Leartsakulpanich
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
mesh_terms: D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008288:Malaria / Q000469:parasitology* / Q000517:prevention & control*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D010966:Plasmodium vivax / Q000201:enzymology*; D013045:Species Specificity; D013940:Thymidylate Synthase / Q000737:chemistry*
chemical_list: D001120:Arginine; D013940:Thymidylate Synthase; D003545:Cysteine