Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit.
DNA gyrase, a typical type II topoisomerase that can introduce negative supercoils in DNA, is essential for replication and transcription in prokaryotes. The apicomplexan parasite Plasmodium falciparum contains the genes for both gyrase A and gyrase B in its genome. Due to the large sizes of both proteins and the unusual codon usage of the highly AT-rich P. falciparum gyrA (PfgyrA) and PfgyrB genes, it has so far been impossible to characterize these proteins, which could be excellent drug targets. Here, we report the cloning, expression, and functional characterization of full-length PfGyrB and functional domains of PfGyrA. Unlike Escherichia coli GyrB, PfGyrB shows strong intrinsic ATPase activity and follows a linear pattern of ATP hydrolysis characteristic of dimer formation in the absence of ATP analogues. These unique features have not been reported for any known gyrase so far. The PfgyrB gene complemented the E. coli gyrase temperature-sensitive strain, and, together with the N-terminal domain of PfGyrA, it showed typical DNA cleavage activity. Furthermore, PfGyrA contains a unique leucine heptad repeat that might be responsible for dimerization. These results confirm the presence of DNA gyrase in eukaryotes and confer great potential for drug development and organelle DNA replication in the deadliest human malarial parasite, P. falciparum.
Dar
Mohd Ashraf
MA
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.
Sharma
Atul
A
Mondal
Neelima
N
Dhar
Suman Kumar
SK
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D053837:DNA Cleavage; D027081:DNA Gyrase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D021122:Protein Subunits / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase