Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase.
Thymidylate 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 chi2913RecA(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 k(cat) value of the R470D+C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Km values 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.
Chanama
Manee
M
Department of Microbiology, Faculty of Public Health, Mahidol University, Rajvithi Road, Bangkok 10400, Thailand.
Chitnumsub
Penchit
P
Yuthavong
Yongyuth
Y
D000818:Animals; D001665:Binding Sites; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D019281:Dimerization; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D005816:Genetic Complementation Test; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D009097:Multienzyme Complexes; D021122:Protein Subunits; C056930:thymidylate synthase-dihydrofolate reductase; C010920:dihydrofolate; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase