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)

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.
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PMID:Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase. 128 74

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.
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PMID:Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase. 138 67

Resistance determinants to the toxic oxyanionic salts of arsenic and antimony are found on plasmids of both gram-negative and gram-positive organisms. In most cases these provide resistance to both the oxyanions of +III oxidation state, antimonite and arsenite, and the +V oxidation state, arsenate. In both gram-positive and -negative bacteria, resistance is correlated with efflux of the anions from cells. The determinant from the plasmid R773, isolated from a gram-negative organism, has been studied in detail. It encodes an oxyanion-translocating ATPase with three subunits, a catalytic subunit, the ArsA protein, a membrane subunit, the ArsB subunit, and a specificity factor, the ArsC protein. The first two form a membrane-bound complex with arsenite-stimulated ATPase activity. The determinants from gram-positive bacteria have only the arsB and arsC genes and encode an efflux system without the participation of an ArsA homologue.
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PMID:Plasmid-encoded resistance to arsenic and antimony. 153 41

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.
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PMID:Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. 153 22

R-factor mediated bacterial resistance to arsenical salts occurs by active extrusion of the toxic oxyanions from cells of gram negative bacteria. The ars operon of the conjugative plasmid R773 encodes an anion pump. The pump has two polypeptide components. The catalytic subunit, the ArsA protein, is an oxyanion-stimulated ATPase. The membrane component, the ArsB protein, has been localized in the inner membrane of Escherichia coli. The ArsA and ArsB proteins have been postulated to form a membrane complex which functions as an anion-translocating ATPase. In this study evidence is presented showing that expression of the arsB gene is required to anchor the ArsA protein to the inner membrane. Binding studies with purified ArsA to membranes with and without the arsB gene product confirm this requirement. Membranes of uncA mutants containing both the ArsA and ArsB proteins exhibit arsenite(antimonite)-stimulated ATPase activity. These results support the model in which the ArsA protein is the catalytic energy transducing component of the anion pump, whereas the integral membrane ArsB protein serves as both the anion channel and membrane binding site for the ArsA protein.
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PMID:Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein. 168 27

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with [alpha-32P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.
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PMID:Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase. 169 16

The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.
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PMID:Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase. 182 84

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with [alpha-32P]ATP. From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. Purified ArsA protein bound a fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine- 5'-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA. Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein.
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PMID:Trinitrophenyl-ATP binding to the ArsA protein: the catalytic subunit of an anion pump. 183 38

The ars (arsenical resistance) operon cloned from R-factor R773 has five genes that encode two repressor proteins, ArsR and ArsD, and three structural proteins, ArsA, ArsB, and ArsC. The ArsA and ArsB proteins form a membrane-bound pump that functions as an oxyanion-translocating ATPase. The substrates of the pump are the oxyanions arsenite or antimonite. The ArsC protein is an arsenate reductase that reduces arsenate to arsenite, which is subsequently pumped out of the cell. This review deals with the mechanism of transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA protein, the catalytic subunit of the pump. The chemical nature of the inducer plays an important role in regulation. In solution arsenite or antimonite exist as oxyanions and reacts with the cysteines in proteins. In both transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA ATPase, the ability of As(III) and Sb(III) to interact with the cysteines of the proteins, involves their action as effector.
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PMID:Mechanisms of metalloregulation of an anion-translocating ATPase. 762 56

Arsenic ions, frequently present as environmental pollutants, are very toxic for most microorganisms. Some microbial strains possess genetic determinants that confer resistance. In bacteria, these determinants are often found on plasmids, which has facilitated their study at the molecular level. Bacterial plasmids conferring arsenic resistance encode specific efflux pumps able to extrude arsenic from the cell cytoplasm thus lowering the intracellular concentration of the toxic ions. In Gram-negative bacteria, the efflux pump consists of a two-component ATPase complex. ArsA is the ATPase subunit and is associated with an integral membrane subunit, ArsB. Arsenate is enzymatically reduced to arsenite (the substrate of ArsB and the activator of ArsA) by the small cytoplasmic ArsC polypeptide. In Gram-positive bacteria, comparable arsB and arsC genes (and proteins) are found, but arsA is missing. In addition to the wide spread plasmid arsenic resistance determinant, a few bacteria confer resistance to arsenite with a separate determinant for enzymatic oxidation of more-toxic arsenite to less-toxic arsenate. In contrast to the detailed information on the mechanisms of arsenic resistance in bacteria, little work has been reported on this subject in algae and fungi.
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PMID:Resistance to arsenic compounds in microorganisms. 784 59


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