Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.
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PMID:The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release. 1135 71

The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.
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PMID:gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity. 1139 91

We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. guilliermondii and C. lusitaniae. Evolutionary relationships among nine Candida species including those of C. albicans and C. glabrata were studied based on the DNA topoisomerase II gene. The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis. Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C. tropicalis and C. parapsilosis are too diverse to be in a single species. A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene. Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.
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PMID:Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species. 1147 May 34

DNA gyrase forms an A(2)B(2) tetramer involved in DNA replication, repair, recombination, and transcription in which the B subunit catalyzes ATP hydrolysis. The Thermus thermophilus and Escherichia coli gyrases are homologues and present the same catalytic activity. When compared with that of the E. coli 43K-5'-adenylyl-beta,gamma-imidodiphosphate complex, the crystal structure of Gyrase B 43K ATPase domain in complex with novobiocin, one of the most potent inhibitors of gyrase shows large conformational changes of the subdomains within the dimer. The stabilization of loop 98-118 closing the active site through dimeric contacts and interaction with domain 2 allows to observe novobiocin-protein interactions that could not be seen in the 24K-inhibitor complexes. Furthermore, this loop adopts a position which defines an "open" conformation of the active site in absence of ATP, in contrast with the "closed" conformation adopted upon ATP binding. All together, these results indicate how the subdomains may propagate conformational changes from the active site and provide crucial information for the design of more specific inhibitors.
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PMID:An open conformation of the Thermus thermophilus gyrase B ATP-binding domain. 1185 Apr 22

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s(-1), respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.
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PMID:Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase. 1200 Aug 34

We have isolated two overlapping genomic clones that contain the 5'-terminal portion of the human vacuolar H(+)-ATPase c subunit (ATP6L) gene. The sequence preceding the transcription initiation site, which is GC-rich, contains four GC boxes and one Oct1-binding site, but there is no TATA box or CCAAT box. In vivo footprint analysis in human cancer cells shows that two GC boxes and the Oct1-binding site are occupied by Sp1 and Oct1, respectively. We show here that treatment with anticancer agents enhances ATP6L expression. Although cisplatin did not induce ATP6L promoter activity, it altered ATP6L mRNA stability. On the other hand, the DNA topoisomerase II inhibitor, TAS-103, strongly induced promoter activity, and this effect was completely eradicated when a mutation was introduced into the Oct1-binding site. Treatment with TAS-103 increased the levels of both Sp1/Sp3 and Oct1 in nuclear extracts. Cooperative binding of Sp1 and Oct1 to the promoter is required for promoter activation by TAS-103. Incubation of a labeled oligonucleotide probe encompassing the -73/-68 GC box and -64/-57 Oct1-binding site with a nuclear extract from drug-treated KB cells yielded higher levels of the specific DNA-protein complex than an extract of untreated cells. Thus, the two transcription factors, Sp1 and Oct1 interact, in an adaptive response to DNA damage, by up-regulating expression of the vacuolar H(+)-ATPase genes. Furthermore, combination of the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A1, with TAS-103 enhanced apoptosis of KB cells with an associated increase in caspase-3 activity. Our data suggest that the induction of V-ATPase expression is an anti-apoptotic defense, and V-ATPase inhibitors in combination with low-dose anticancer agents may provide a new therapeutic approach.
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PMID:Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents. 1213 27

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.
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PMID:The ATP-binding site of type II topoisomerases as a target for antibacterial drugs. 1257 Jul 64

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.
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PMID:Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance. 1260 39

The GHKL phosphotransferase superfamily, characterized by four sequence motifs that form the ATP-binding site, consists of the ATPase domains of type II DNA topoisomerases, Hsp90, and MutL, and bacterial and mitochondrial protein kinases. In addition to a magnesium ion, which is essential for catalysis, a potassium ion bound adjacent to the triphosphate moiety of ATP in a rat mitochondrial protein kinase, BCK (branched-chain alpha-ketoacid dehydrogenase kinase), has been shown to be indispensable for nucleotide binding and hydrolysis. Using X-ray crystallographic, biochemical, and genetic analyses, we find that the monovalent cation-binding site is conserved in MutL, but both Na(+) and K(+) support the MutL ATPase activity. When Ala100 of MutL is substituted by proline, mimicking the K(+)-binding environment in BCK, the mutant MutL protein becomes exclusively dependent on Na(+) for the ATPase activity. The coordination of this Na(+) ion is identical to that of the K(+) ion in BCK and involves four carbonyl oxygen atoms emanating from the hinges of the ATP lid and a non-bridging oxygen of the bound nucleotide. A similar monovalent cation-binding site is found in DNA gyrase with additional coordination by a serine side chain. The conserved and protein-specific monovalent cation-binding site is unique to the GHKL superfamily and probably essential for both ATPase and kinase activity. Dependence on different monovalent cations for catalysis may be exploited for future drug design specifically targeting each individual member of the GHKL superfamily.
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PMID:Monovalent cation dependence and preference of GHKL ATPases and kinases. 1278 29

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl- N-nitro- N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.
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PMID:Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents. 1368 98


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