Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum.
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.
Kasekarn
Waraporn
W
Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Sirawaraporn
Rachada
R
Chahomchuen
Thippayarat
T
Cowman
Alan F
AF
Sirawaraporn
Worachart
W
D000255:Adenosine Triphosphate / Q000494:pharmacology; D010131:Aminosalicylic Acid / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D003622:Dapsone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013424:Sulfanilamides / Q000494:pharmacology; D013696:Temperature
C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D003067:Coenzymes; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D013424:Sulfanilamides; D010131:Aminosalicylic Acid; D000255:Adenosine Triphosphate; D003622:Dapsone; D008274:Magnesium