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PubMed1996

Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump.

The ArsA ATPase is the catalytic subunit of the Ars pump that catalyzes arsenical extrusion in Escherichia coli, thus providing resistance. The active form of ArsA is a homodimer with four nucleotide binding sites, two from each monomer. The codons for Gly-15 in the N-terminal consensus nucleotide binding sequence and Gly-334 in the C-terminal sequence were individually mutated to cysteine codons. Cells expressing an arsAG334C mutation retained arsenite resistance, while an arsAG15C mutation resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. Selection for suppression of the G15C mutation that restored arsenite resistance yielded an A344V substitution. Ala-344 is located adjacent to the C-terminal nucleotide binding sequence. The second site mutation did not suppress the loss of resistance resulting from G18D, G20S, or T22I substitutions in the N-terminal nucleotide binding site. Cells expressing the G15C/A344V double mutant regained arsenite extrusion. These results suggest a spatial proximity of Gly-15 and Ala-344 and support a model for interaction of the nucleotide binding sites in ArsA.

Adenosine Triphosphatases↗
PubMed1998

Steric limitations in the interaction of the ATP binding domains of the ArsA ATPase.

ArsA, the catalytic subunit of an anion-translocating ATPase, has two consensus nucleotide binding sites, one N-terminal and one C-terminal. A mutation producing a G15C substitution in the N-terminal domain resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. A second site revertant (A344V) adjacent to the C-terminal nucleotide binding site was previously shown to restore arsenite resistance, suggesting the interaction of the nucleotide binding sites in ArsA (Li, J., Liu, S., and Rosen, B. P. (1996) J. Biol. Chem. 271, 25247-25252). In this study, it is shown that alteration of Ala-344 to bulkier residues, including Cys, Thr, Pro, Asp, Leu, Phe, Tyr, or Arg, also suppressed the G15C substitution. However, A344G or A344S substitutions only marginally suppressed the primary mutation. Alteration of Gly-15 to Ala, Cys, Asp, Tyr, or Arg each resulted in decreased arsenite resistance. The larger the residue volume of the substitution, the lower the resistance, with a G15R substitution producing the least resistance. Resistance in a strain expressing an arsA gene encoding the G15R substitution could be rescued by A344S, A344T, A344D, A344R, or A344V second site suppressors. The larger the residue is then the greater the suppression is. The in vitro ArsA ATPase activities from purified wild type, G15A, G15C, and G15R exhibits an inverse relationship between activity and residue volume. Purified G15A and G15C exhibited both an increase in the Km for ATP and a decrease in Vmax. The results are consistent with a physical interaction of the two nucleotide binding domains and indicate that the geometry at the interface between the N- and C-terminal nucleotide binding sites places spatial constraints on allowable residues in that interface.

Adenosine Triphosphatases↗
PubMed1999

Families of arsenic transporters.

Bacterial arsenic resistance (ars) operons encode an arsenite-efflux system that can be a secondary carrier protein (ArsB) or an anion-translocating ATPase (ArsAB). Yeasts extrude arsenite using Acr3p, a plasma membrane carrier protein, or sequester it in vacuoles as the glutathione conjugate using Ycf1p, an ABC transporter.

ATP-Binding Cassette Transporters↗
PubMed1998

Metalloid resistance mechanisms in prokaryotes.

Resistance to antibiotics and other chemotherapeutic agents is becoming a wide spread health issue. The biochemical mechanisms of resistance vary, but active efflux of the toxic agents is one of the most common. Bacterial resistances to metals provide good model systems for transport-related resistances. One of the best understood metal resistance systems is the product of the ars operon, which provides resistance to arsenicals and antimonials. As a reflection of the ubiquity of arsenic in the environment, ars operons are found in all species of bacteria, carried in chromosomes, plasmids, and transposons. This review focuses on the biochemistry of the proteins of the ars operon of R-factor R773. The system is novel in several respects. First, it is regulated at the transcriptional and allosteric levels, and regulation is effected through cysteine thiol interaction with As(III) or Sb(III). Thus soft metal-thiol chemistry provides a high affinity digital switch to turn the regulated protein on with rapidity. The transport system that provides resistance, on the other hand, uses oxyanions of arsenic or antimony as substrates. This nonmetal chemistry allows for low affinity interactions of the membrane transporter with substrate, conductive with translocation and release of substrate on the outside of the cell membrane. Second, the transporter is uniquely capable of coupling to either electrochemical energy as a secondary carrier protein or the chemical energy of ATP when binding of a catalytic subunit converts it into an anion-translocating ATPase.

Adenosine Triphosphatases↗
PubMed1994

In vitro assembly of an anion-stimulated ATPase from peptide fragments.

The oxyanion-translocating ATPase encoded by the ars operon of plasmid R 773 confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the oxyanions from the cells. The catalytic subunit, the 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. In this report subclones of the arsA gene were constructed to produce polypeptide fragments of the ArsA protein. By themselves none of the fragments exhibited anion-stimulated ATPase activity. Denaturation and renaturation of mixtures of N- and C-terminal polypeptides that between them comprised an entire ArsA protein resulted in active ATPase complexes.

Adenosine Triphosphatases↗
PubMed1996

Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter.

Arsenite resistance in bacteria is mediated by an efflux pump composed of the arsA and arsB gene products. We have isolated the human homolog of the bacterial arsA (hARSA-I), a member of the ATPase superfamily with no transmembrane domain. Southern and Northern analyses indicated the presence of two cross-hybridizing genes in the human genome and expression of hARSA-I in many tissues. A rabbit antiserum raised against a glutathione-S-transferase (GST)/hARSA-I fusion protein identified two cross-reacting proteins of 37 and 42 kDa by Western analysis in two different human cell lines. Overexpression of hARSA-I in the embryonal human kidney 293 cell line was accompanied by overproduction of the 37-kDa protein Biochemical analysis using the GST/hARSA-I fusion protein indicated that hARSA-I is an ATPase analogous to the bacterial ArsA. Thus, hARSA-I is a new eukaryotic member of a highly conserved ATP-binding superfamily of proteins.

Adenosine Triphosphatases↗
PubMed1998

Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine.

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.

Adenosine↗
PubMed2003

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes.

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.

Amino Acid Sequence↗
PubMed1994

Interaction of the catalytic and the membrane subunits of an oxyanion-translocating ATPase.

Resistance to arsenical and antimonial compounds in Escherichia coli is due to active extrusion of these compounds from cells expressing the ars operon. The arsenical pump is an ion-translocating ATPase which consists of two polypeptide components, the ArsA and ArsB proteins. The ArsB protein, the inner membrane component of the pump, has been shown to function as the membrane anchor for the catalytic subunit, the ArsA protein. The properties and nature of interaction between these two components of the pump were investigated using an in vitro binding assay. Purified ArsA protein bound to the membrane in a saturable manner. In the absence of arsenite or antimonite an apparent positive cooperativity in the binding of the ArsA protein to membrane vesicles containing the ArsB protein was observed. In the presence of arsenite or antimonite binding became hyperbolic, with a 10-fold decrease in the concentration of ArsA protein required for half-maximal binding, without any change in the stoichiometry of the complex. Addition of ATP had little affect on membrane binding of the ArsA ATPase subunit. In the presence or absence of the anionic substrates binding was maximal in a pH range 7.5-8.5.

Adenosine Triphosphatases↗
PubMed1995

Mechanisms of metalloregulation of an anion-translocating ATPase.

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.

Adenosine Triphosphatases↗
PubMed1992

Plasmid-encoded resistance to arsenic and antimony.

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.

Adenosine Triphosphatases↗
PubMed1991

Substrate-induced dimerization of the ArsA protein, the catalytic component of an anion-translocating ATPase.

The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite, suggesting that the binding of antimonite increased the affinity of ArsA protein for FSBA. Incorporation of [14C]FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.

Adenosine↗
PubMed1992

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

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.

Adenosine Triphosphatases↗
PubMed2003

MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation.

The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved 'signature' lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.

Adenosine Triphosphatases↗
PubMed1994

Solute transport and energy transduction in bacteria.

In bacteria two forms of metabolic energy are usually present, i.e. ATP and transmembrane ion-gradients, that can be used to drive the various endergonic reactions associated with cellular growth. ATP can be formed directly in substrate level phosphorylation reactions whereas primary transport processes can generate the ion-gradients across the cytoplasmic membrane. The two forms of metabolic energy can be interconverted by the action of ion-translocating ATPases. For fermentative organisms it has long been thought that ion-gradients could only be generated at the expense of ATP hydrolysis by the F0F1-ATPase. In the present article, an overview is given of the various secondary transport processes that form ion-gradients at the expense of precursor (substrate) and/or end-product concentration gradients. The metabolic energy formed by these chemiosmotic circuits contributes to the 'energy status' of the bacterial cell which is particularly important for anaerobic/fermentative organisms.

Adenosine Triphosphatases↗
PubMed2001

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump.

The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.

Adenosine Triphosphatases↗
PubMed1998

Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I).

The arsenite-stimulated human ATPase (hASNA-I) protein is a distinct human ATPase whose cDNA was cloned by sequence homology to the Escherichia coli ATPase arsA. Its subcellular localization in human malignant melanoma T289 cells was examined to gain insight into the role of hASNA-I in the physiology of human cells. Immunocytochemical staining using the specific anti-hASNA-I monoclonal antibody 5G8 showed a cytoplasmic, perinuclear, and nucleolar distribution. Subcellular fractionation indicated that the cytoplasmic hASNA-I was soluble and that the perinuclear distribution was due to association with the nuclear membrane rather than with the endoplasmic reticulum. Its presence in the nucleolus was confirmed by showing colocalization with an antibody of known nucleolar specificity. Further immunocytochemical analysis showed that the hASNA-I at the nuclear membrane was associated with invaginations into the nucleus in interphase cells. These results indicate that hASNA-I is a paralogue of the bacterial ArsA protein and suggest that it plays a role in the nucleocytoplasmic transport of a nucleolar component.

Adenosine Triphosphatases↗