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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/21156
Title: Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum
Authors: Waraporn Kasekarn
Rachada Sirawaraporn
Thippayarat Chahomchuen
Alan F. Cowman
Worachart Sirawaraporn
Mahidol University
Walter Elisa Hall Inst. of Med. Res.
Keywords: Biochemistry, Genetics and Molecular Biology;Immunology and Microbiology
Issue Date: 1-Sep-2004
Citation: Molecular and Biochemical Parasitology. Vol.137, No.1 (2004), 43-53
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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=3342978121&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/21156
ISSN: 01666851
Appears in Collections:Scopus 2001-2005

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