Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum

Mol Biochem Parasitol. 2004 Sep;137(1):43-53. doi: 10.1016/j.molbiopara.2004.04.012.

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 approximately 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 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Aminosalicylic Acid / pharmacology
  • Animals
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cloning, Molecular
  • Coenzymes / pharmacology
  • Dapsone / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Hydrogen-Ion Concentration
  • Magnesium / pharmacology
  • Molecular Weight
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Plasmodium falciparum / enzymology*
  • Plasmodium falciparum / genetics
  • Protein Subunits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sulfanilamides / pharmacology
  • Temperature

Substances

  • 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase
  • Coenzymes
  • Enzyme Inhibitors
  • Multienzyme Complexes
  • Protein Subunits
  • Recombinant Proteins
  • Sulfanilamides
  • Aminosalicylic Acid
  • Adenosine Triphosphate
  • Dapsone
  • Magnesium