Article
Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum.
BIOTEC, National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand.
Acta Crystallographica Section D Biological Crystallography (impact factor:
12.62).
05/2004;
60(Pt 4):780-3.
DOI:10.1107/S0907444904001544
pp.780-3
Source: PubMed
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Citations (0)
- Cited In (6)
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Article: Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.
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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.Parasitology International 06/2012; 61(2):324-32. · 2.13 Impact Factor -
Article: Formation of catalytically active cross-species heterodimers of thymidylate synthase from Plasmodium falciparum and Plasmodium vivax.
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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.Molecular Biology Reports 02/2011; 38(2):1029-37. · 2.93 Impact Factor -
Article: Trypanosomal dihydrofolate reductase reveals natural antifolate resistance.
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ABSTRACT: Dihydrofolate reductase (DHFR) is a potential drug target for Trypanosoma brucei, a human parasite, which is the causative agent for African sleeping sickness. No drug is available against this target, since none of the classical antifolates such as pyrimethamine (PYR), cycloguanil, or trimethoprim are effective as selective inhibitors of T. brucei DHFR (TbDHFR). In order to design effective drugs that target TbDHFR, co-crystal structures with bound antifolates were studied. On comparison with malarial Plasmodium falciparum DHFR (PfDHFR), the co-crystal structures of wild-type TbDHFR reveal greater structural similarities to a mutant PfDHFR causing antifolate resistance than the wild-type enzyme. TbDHFR imposes steric hindrance for rigid inhibitors like PYR around Thr86, which is equivalent to Ser108Asn of the malarial enzymes. In addition, a missing residue on TbDHFR active-site loop together with the presence of Ile51 widens its active site even further than the structural effect of Asn51Ile, which is observed in PfDHFR structures. The structural similarities are paralleled by the similarly poor affinities of the trypanosomal enzyme for rigid inhibitors. Mutations of TbDHFR at Thr86 resulted in 10-fold enhancement or 7-fold reduction in the rigid inhibitors affinities for Thr86Ser or Thr86Asn, respectively. The co-crystal structure of TbDHFR with a flexible antifolate WR99210 suggests that its greater affinity result from its ability to avoid such Thr86 clash and occupy the widened binding space similarly to what is observed in the PfDHFR structures. Natural resistance to antifolates of TbDHFR can therefore be explained, and potential antifolate chemotherapy of trypanosomiasis should be possible taking this into account.ACS Chemical Biology 06/2011; 6(9):905-11. · 6.45 Impact Factor
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Keywords
52% solvent content
asymmetric unit
baby oil
Conditions
corresponding Plasmodium falciparum parasite
cryogenic conditions
double mutant K1CB1
Escherichia coli BL21
full-length pfdhfr-ts genes
microbatch setup
modified microbatch method
modified pET(17b)
P. falciparum dihydrofolate reductase-thymidylate synthase
particular type
PfDHFR-TS crystallization
Preliminary X-ray analysis
purified enzymes
soluble active enzyme
two PfDHFR-TS proteins
X-ray diffraction data
