Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli.
Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.
Jayalakshmi
R
R
Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560 064, India.
Sumathy
K
K
Balaram
Hemalatha
H
D000265:Adenylosuccinate Synthase / Q000096:biosynthesis* / Q000737:chemistry*; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005816:Genetic Complementation Test; D005990:Glycerol / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007291:Inosine Monophosphate / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007291:Inosine Monophosphate; D001224:Aspartic Acid; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D000265:Adenylosuccinate Synthase; D005990:Glycerol