Publication: Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum
Issued Date
2004-09-01
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ISSN
01666851
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2-s2.0-3342978121
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Mahidol University
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SCOPUS
Bibliographic Citation
Molecular and Biochemical Parasitology. Vol.137, No.1 (2004), 43-53
Suggested Citation
Waraporn Kasekarn, Rachada Sirawaraporn, Thippayarat Chahomchuen, Alan F. Cowman, Worachart Sirawaraporn Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum. Molecular and Biochemical Parasitology. Vol.137, No.1 (2004), 43-53. doi:10.1016/j.molbiopara.2004.04.012 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21156
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Title
Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum
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Abstract
A 2118-base pair gene encoding the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses of Plasmodium falciparum (pfPPPK-DHPS) was expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain. The enzyme was purified to near homogeneity using nickel affinity chromatography followed by gel filtration and migrates as an intense band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of ∼83 kDa. Gel filtration suggested that the native pfPPPK-DHPS might exist as a tetramer of identical subunits. The enzyme was found to be Mg2+- and ATP-dependent and had optimal temperature ranging from 37 to 45°C with peak activity at pH 10. Sodium chloride and potassium chloride at 0.2 and 0.4 M, respectively, activated the activity of the enzyme but higher salt concentrations were inhibitory. Guanidine-HCl and urea inhibited the enzyme activity by 50% at 0.25 and 0.9 M, respectively. Kinetic properties of the recombinant pfPPPK-DHPS were investigated. Sulfathiazole and dapsone were potent inhibitors of pfPPPK-DHPS, whilst sulfadoxine, sulfanilamide, sulfacetamide and p-aminosalicylic acid were less inhibitory. Our construct provides an abundant source of recombinant pfPPPK-DHPS for crystallization and drug screening. © 2004 Elsevier B.V. All rights reserved.