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B P Rosen

Publications and source records attributed to B P Rosen.

At least 19 recordsLinked to original sources
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↗
PubMed2001

Escherichia coli CopA N-terminal Cys(X)(2)Cys motifs are not required for copper resistance or transport.

Escherichia coli CopA is a Cu(I)-translocating P-type ATPase that is involved in copper export and resistance. It is an orthologue of the human Menkes and Wilson disease-related proteins. Each of those two human copper pumps has six N-terminal Cys(X)(2)Cys sequences, but their function in transport is unclear. CopA has two N-terminal Cys(X)(2)Cys sequences, GLSC(14)GHC(17) and GMSC(110)ASC(113). The requirement of these cysteine motifs was investigated by mutagenesis of the codons for all four cysteine residues, singly and in combination. Cells of a copA deletion strain expressing genes for the mutant genes were nearly as resistant to copper as the wild type. In addition, everted membrane vesicles from cells expressing the mutant copA genes exhibited ATP-coupled accumulation of copper similar to that of the wild type. The results indicate that neither of two N-terminal Cys(X)(2)Cys motifs is required for either resistance or transport.

Adenosine Triphosphate↗
PubMed2001

The phosphatase C(X)5R motif is required for catalytic activity of the Saccharomyces cerevisiae Acr2p arsenate reductase.

Acr2p detoxifies arsenate by reduction to arsenite in Saccharomyces cerevisiae. This reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. Acr2p has a HC(X)(5)R motif, the signature sequence of the phosphate binding loop of the dual-specific and protein-tyrosine phosphatase family. In Acr2p these are residues His-75, Cys-76, and Arg-82, respectively. Acr2p has another sequence, (118)HCR, that is absent in phosphatases. Acr2p also has a third cysteine residue at position 106. Each of these cysteine residues was changed individually to serine residues, whereas the histidine and arginine residues were altered to alanines. Cells of Escherichia coli heterologously expressing the majority of the mutant ACR2 genes retained wild type resistance to arsenate, and the purified altered Acr2p proteins exhibited normal enzymatic properties. In contrast, cells expressing either the C76S or R82A mutations lost resistance to arsenate, and the purified proteins were inactive. These results suggest that Acr2p utilizes a phosphatase-like Cys(X)(5)Arg motif as the catalytic center to reduce arsenate to arsenite.

Adenosine Triphosphatases↗
PubMed2001

Genomic organization and chromosomal localization of the Asna1 gene, a mouse homologue of a bacterial arsenic-translocating ATPase gene.

The plasmid encoded ArsA ATPase in Escherichia coli is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Arsenite or antimonite allosterically activates the ArsA ATPase activity. In this paper, we report the cloning and characterization of the mouse homologue (Asna1) of the bacterial arsA gene. The Asna1 gene encodes an open reading frame of 348 amino acids and exhibits 27% identity to the bacterial ArsA protein and 99% similarity to its human counterpart (hASNA-1). The Asna1 mRNA is a approximately 1.3 kb transcript and is present at high levels in kidney and testis, moderate levels in brain, liver, lung and skin, and low levels in heart, small intestine, spleen, stomach, and thymus. A negligible amount of Asna1 transcript is detected in skeletal muscle. We have also characterized the genomic structure of the Asna1 gene. The gene spans over 7 kb and consists of seven exons and six introns. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 4 that is GC instead of GT. Fluorescence in situ hybridization indicates that the Asna1 gene is localized in the C3-D1 region of mouse chromosome 8.

Adenosine Triphosphatases↗
PubMed2001

Conformational changes in four regions of the Escherichia coli ArsA ATPase link ATP hydrolysis to ion translocation.

Structures of ArsA with ATP, AMP-PNP, or ADP.AlF(3) bound at the A2 nucleotide binding site were determined. Binding of different nucleotides modifies the coordination sphere of Mg(2+). In particular, the changes elicited by ADP.AlF(3) provide insights into the mechanism of ATP hydrolysis. In-line attack by water onto the gamma-phosphate of ATP would be followed first by formation of a trigonal intermediate and then by breaking of the scissile bond between the beta- and gamma-phosphates. Motions of amino acid side chains at the A2 nucleotide binding site during ATP binding and hydrolysis propagate at a distance, producing conformational changes in four different regions of the protein corresponding to helices H4-H5, helices H9-H10, helices H13-H15, and to the S1-H2-S2 region. These elements are extensions of, respectively, the Switch I and Switch II regions, the A-loop (a small loop near the nucleotide adenine moiety), and the P-loop. Based on the observed conformational changes, it is proposed that ArsA functions as a reciprocating engine that hydrolyzes 2 mol of ATP per each cycle of ion translocation across the membrane.

Adenine↗
PubMed2001

The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase.

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein were fused at the COOH terminus of PGPA. Immunofluorescence, confocal, and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PGPAHA transfectants was performed to further characterize this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmol/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transports thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.

ATP-Binding Cassette Transporters↗
PubMed2001

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.

Allosteric Regulation↗
PubMed2001

Role of cysteinyl residues in sensing Pb(II), Cd(II), and Zn(II) by the plasmid pI258 CadC repressor.

The cadCA operon of Staphylococcus aureus plasmid pI258 confers resistance to salts of the soft metals lead, cadmium, and zinc. The operon is regulated by CadC, a member of the ArsR family of metal-responsive transcriptional repressors. In this study the role of the five cysteine residues of CadC in soft metal ion sensing was investigated. Cys-7, Cys-11, Cys-52, Cys-58, and Cys-60 were changed individually to glycine or serine residues. The effect of the cadC mutations was examined in Escherichia coli using a green fluorescent protein reporter system. None of the mutations affected the ability of CadC to repress gfp expression. Neither Cys-11 nor Cys-52 was required for in vivo response to Pb(II), Zn(II), or Cd(II). Cys-7, Cys-58, or Cys-60 mutations each reduced or eliminated soft metal sensing. Wild-type and mutant CadC proteins were purified, and the effect of the substitutions on DNA binding was determined using a restriction enzyme protection assay. Binding of wild-type CadC protected cad operator DNA from digestion at the single SspI site, and the addition of Pb(II), Zn(II), or Cd(II) resulted in deprotection. Chemical modification of the cysteine residues in CadC had no effect on protection but eliminated deprotection. C11G and C52G proteins exhibited wild-type properties in vitro. C7G, C58S, and C60G proteins were able to be protected from SspI digestion but had reduced responses to soft metal ions. The results indicate that Cys-7, Cys-58, and Cys-60 are involved in sensing those soft metals and suggest that they are ligands to Pb(II), Zn(II), and Cd(II).

Amino Acid Sequence↗
PubMed2001

Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.

BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.

Arsenates↗
PubMed2001

Structure-function analysis of the ArsA ATPase: contribution of histidine residues.

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.

Antimony↗
PubMed2001

Role of vicinal cysteine pairs in metalloid sensing by the ArsD As(III)-responsive repressor.

ArsD is a 120-residue repressor that regulates expression of the arsRDABC arsenical resistance operon of plasmid R773 in Escherichia coli. ArsD is released from arsRDABC promoter DNA by binding of the compounds with the metalloids As(III) or Sb(III). ArsD has three vicinal cysteine pairs, Cys-12 and Cys-13, Cys-112 and Cys-113 and Cys-119 and Cys-120. In this study, the role of these three cysteine pairs was investigated. Mutation or deletion of Cys-119-Cys-120 had no effect on repression or metalloid responsiveness in vivo or in vitro. Mutagenesis of either the Cys-12-Cys-13 pair or the Cys-112-Cys-113 pair had no effect on repression but produced loss of inducibility, suggesting that both Cys-12-Cys-13 and Cys-112-Cys-113 may be required for As(III) or Sb(III) responsiveness. Assays of binding of wild-type and mutant ArsDs by As(III) affinity chromatography showed that each of the three vicinal cysteine pairs is capable of binding As(III) independently. The effect of As(III) or Sb(III) on intrinsic protein fluorescence was used to examine the properties of individual cysteine pairs. The fluorescence of Trp-97 was shown to be quenched by the addition of Sb(III) or As(III). The vicinal Cys-112-Cys-113 pair was required for the majority of the metalloid-dependent quenching of Trp-97 fluorescence. The data are consistent with a model in which Cys-12-Cys-13 and Cys-112-Cys-113 form independent As(III) binding sites, both of which are required for in vivo ArsD function.

Amino Acid Sequence↗
PubMed2001

NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance.

There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance.

Alcaligenes↗
PubMed2000

A kinetic model for the action of a resistance efflux pump.

ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting NBD, for which the structure is known. On the basis of a rigorous analysis of the pre-steady-state kinetics of nucleotide binding and hydrolysis, we propose a model in which ArsA alternates between two mutually exclusive conformations as follows: the ArsA(1) conformation in which the A1 site is closed but the A2 site open; and the ArsA(2) conformation, in which the A1 and A2 sites are open and closed, respectively. Antimonite elicits its effects by sequestering ArsA in the ArsA(1) conformation, which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes this process to sequester Sb(III) from the medium and eject it into the channel of ArsB.

Adenosine Diphosphate↗
PubMed2000

Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump.

Active extrusion is a common mechanism underlying detoxification of heavy metals, drugs and antibiotics in bacteria, protozoa and mammals. In Escherichia coli, the ArsAB pump provides resistance to arsenite and antimonite. This pump consists of a soluble ATPase (ArsA) and a membrane channel (ArsB). ArsA contains two nucleotide-binding sites (NBSs) and a binding site for arsenic or antimony. Binding of metalloids stimulates ATPase activity. The crystal structure of ArsA reveals that both NBSs and the metal-binding site are located at the interface between two homologous domains. A short stretch of residues connecting the metal-binding site to the NBSs provides a signal transduction pathway that conveys information on metal occupancy to the ATP hydrolysis sites. Based on these structural features, we propose that the metal-binding site is involved directly in the process of vectorial translocation of arsenite or antimonite across the membrane. The relative positions of the NBS and the inferred mechanism of allosteric activation of ArsA provide a useful model for the interaction of the catalytic domains in other transport ATPases.

Adenosine Triphosphatases↗
PubMed2000

Purification and characterization of ACR2p, the Saccharomyces cerevisiae arsenate reductase.

In Saccharomyces cerevisiae, expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2 complemented the arsenate-sensitive phenotype of an arsC deletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis of grx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase.

Adenosine Triphosphatases↗
PubMed2000

The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli.

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

Adenosine Triphosphatases↗