Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum
- Author(s)
- Kasekarn, W; Sirawaraporn, R; Chahomchuen, T; Cowman, AF; Sirawaraporn, W;
- Details
- Publication Year 2004-09,Volume 137,Issue #1,Page 43-53
- Journal Title
- MOLECULAR AND BIOCHEMICAL PARASITOLOGY
- Publication Type
- Journal Article
- 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 similar to83 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 degreesC 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. (C) 2004 Elsevier B.V. All rights reserved.
- Publisher
- ELSEVIER SCIENCE BV
- Keywords
- HUMAN MALARIA PARASITE; ESCHERICHIA-COLI; FOLIC-ACID; SULFONAMIDE RESISTANCE; STREPTOCOCCUS-PNEUMONIAE; SULFADOXINE RESISTANCE; CRYSTAL-STRUCTURE; SULFA DRUGS; GENE; SYNTHETASE
- Publisher's Version
- https://doi.org/10.1016/j.molbiopara.2004.04.012
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- Refer to copyright notice on published article.
Creation Date: 2004-09-01 12:00:00