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Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit.

ABSTRACT: 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.

SUBMITTER: Dar MA

PROVIDER: S-EPMC1828931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors.ResultsWe have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures.ConclusionThe apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a β-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation and development of inhibitors for apicoplast gyrases.

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Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit.

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