scholarly journals Molecular Cloning of Apicoplast-Targeted Plasmodium falciparum DNA Gyrase Genes: Unique Intrinsic ATPase Activity and ATP-Independent Dimerization of PfGyrB Subunit

2007 ◽  
Vol 6 (3) ◽  
pp. 398-412 ◽  
Author(s):  
Mohd Ashraf Dar ◽  
Atul Sharma ◽  
Neelima Mondal ◽  
Suman Kumar Dhar
Keyword(s):  
Plasmodium Falciparum ◽  
Atpase Activity ◽  
Dna Cleavage ◽  
Functional Characterization ◽  
Dna Gyrase ◽  
Heptad Repeat ◽  
Malarial Parasite ◽  
Temperature Sensitive ◽  
Linear Pattern ◽  
Gyrase A

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.

scholarly journals A Unique 45-Amino-Acid Region in the Toprim Domain of Plasmodium falciparum Gyrase B Is Essential for Its Activity

2009 ◽  
Vol 8 (11) ◽  
pp. 1759-1769 ◽  
Author(s):  
Ashraf Dar ◽  
Dhaneswar Prusty ◽  
Neelima Mondal ◽  
Suman K. Dhar
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Dna Binding ◽  
Atpase Activity ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Binding Region ◽  
Amino Acid Region ◽  
Acid Region

ABSTRACT DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction.

scholarly journals The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic

2021 ◽  
Vol 118 (11) ◽  
pp. e2016705118
Author(s):  
Lipeng Feng ◽  
Julia E. A. Mundy ◽  
Clare E. M. Stevenson ◽  
Lesley A. Mitchenall ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Dna Gyrase ◽  
Dna Breaks ◽  
Repeat Protein ◽  
X Ray Crystallography ◽  
Gyrase A ◽  
Negative Supercoiling

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

scholarly journals Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser83and Asp87

2001 ◽  
Vol 45 (7) ◽  
pp. 1994-2000 ◽  
Author(s):  
Faye M. Barnard ◽  
Anthony Maxwell
Keyword(s):  
Dna Cleavage ◽  
Double Mutant ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Drug Binding ◽  
Resistance Mutations ◽  
Mutant Proteins ◽  
Gyrase A ◽  
A Subunit ◽  
High Level

ABSTRACT DNA gyrase is a target of quinolone antibacterial agents, but the molecular details of the quinolone-gyrase interaction are not clear. Quinolone resistance mutations frequently occur at residues Ser83 and Asp87 of the gyrase A subunit, suggesting that these residues are involved in drug binding. Single and double alanine substitutions were created at these positions (Ala83, Ala87, and Ala83Ala87), and the mutant proteins were assessed for DNA supercoiling, DNA cleavage, and resistance to a number of quinolone drugs. The Ala83 mutant was fully active in supercoiling, whereas the Ala87 and the double mutant were 2.5- and 4- to 5-fold less active, respectively; this loss in activity may be partly due to an increased affinity of these mutant proteins for DNA. Supercoiling inhibition and cleavage assays revealed that the double mutant has a high level of resistance to certain quinolones while the mutants with single alanine substitutions show low-level resistance. Using a drug-binding assay we demonstrated that the double-mutant enzyme-DNA complex has a lower affinity for ciprofloxacin than the wild-type complex. Based on the pattern of resistance to a series of quinolones, an interaction between the C-8 group of the quinolone and the double-mutant gyrase in the region of residues 83 and 87 is proposed.

scholarly journals Isolation and Functional Characterization of Two Distinct Sexual-Stage-Specific Promoters of the Human Malaria Parasite Plasmodium falciparum

1999 ◽  
Vol 19 (2) ◽  
pp. 967-978 ◽  
Author(s):  
Koen J. Dechering ◽  
Anita M. Kaan ◽  
Wilfred Mbacham ◽  
Dyann F. Wirth ◽  
Wijnand Eling ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Sexual Differentiation ◽  
Functional Characterization ◽  
Malarial Parasite ◽  
Mosquito Vector ◽  
Differentiation Process ◽  
Successful Development ◽  
Sexual Stages ◽  
Developmental Switches

ABSTRACT Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of thepfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activatespfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

scholarly journals Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins.

1984 ◽  
Vol 259 (14) ◽  
pp. 9199-9201 ◽  
Author(s):  
K Mizuuchi ◽  
M Mizuuchi ◽  
M H O'Dea ◽  
M Gellert
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
Gyrase A

scholarly journals Localization of ferrochelatase in Plasmodium falciparum

2004 ◽  
Vol 384 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Sundaramurthy VARADHARAJAN ◽  
B. K. Chandrashekar SAGAR ◽  
Pundi N. RANGARAJAN ◽  
Govindarajan PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Enzymic Activity ◽  
Malarial Parasite ◽  
Parasite Genome ◽  
Marker Proteins ◽  
Parasite Plasmodium ◽  
Theoretical Predictions ◽  
Parasite Plasmodium Falciparum

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

Sign in / Sign up

Export Citation Format

Share Document