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PubMed2002

Biochemical evidence for interaction between the two nucleotide binding domains of ArsA. Insights from mutants and ATP analogs.

ArsA, the peripheral membrane component of the anion-translocating ATPase ArsAB, consists of two nucleotide binding domains (A1 and A2), which are connected by a linker sequence. Previous studies on ArsA have focused on the function of each nucleotide binding domain and the role of the linker, whereas the present study looks at the interactions between the binding domains and their interactions with the linker. It has previously been shown that the A1 domain of ArsA carries out unisite catalysis in the absence of antimonite, while A2 is recruited in multisite catalysis by antimonite in the presence of a functional A1 domain. Multisite catalysis thus seems to result from an interaction between A1 and A2 brought about by antimonite. In the present study, we provide direct biochemical evidence for interaction between the two nucleotide binding domains and show that the linker region acts as a transducer of the conformational changes between them. We find that nucleotide binding to the A2 domain results in a significant, detectable change in the conformation of the A1 domain. Two ATP analogs, FSBA and ATP gamma S, used in this study, were both found to bind preferentially to the A2 domain, and their binding resulted in changing the otherwise compact A1 domain into an open conformation. Point mutations in the A2 domain and the linker region also produced a similar effect on the conformation of A1, thus suggesting that events at A2 are relayed to A1 via the linker. We propose that nucleotide binding to A2 produces a two-tiered conformational change. The significance of these changes in the mechanism of ArsA is discussed.

Adenosine Triphosphatases↗

PubMed2006

The activation of electrophile, nucleophile and leaving group during the reaction catalysed by pI258 arsenate reductase.

The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.

Arsenates↗

PubMed1994

Crystallization and preliminary X-ray diffraction analysis of the ArsC protein from the Escherichia coli arsenical resistance plasmid, R773.

Diffraction data to 3.0 A resolution were collected on crystals of ArsC protein from the conjugative resistance factor R773 which mediates arsenical resistance in the Gram negative bacterium Escherichia coli. The crystal system is tetragonal, a = 116.2 A, c = 145.0 A and the space group is either P4(1)2(1)2 or P4(3)2(1)2. The most probable range for the contents of the asymmetric unit is four to eight ArsC molecules (M(r) = 15,811).

Adenosine Triphosphatases↗

PubMedDate not supplied

A 61-kb ring chromosome shows an ARS-dependent increase in its mitotic stability in the mcm2 mutant of yeast.

We have studied the effects of ARS addition and deletion on the maintenance of a 61-kb ring derivative of chromosome III in a minichromosome maintenance mutant of yeast carrying the mcm2-1 mutation. When this ring chromosome, CIIIR, had either of its two strong origins deleted, the resultant chromosome showed a much greater instability in the mutant as compared to that of the wild-type strain. Integration of more ARSs improved the maintenance of CIIIR in the mutant but not in the wild-type strain. Increase in the size of CIIIR, without any ARS addition, did not improve the stability in either strain. A spontaneous revertant for improved growth at 35 degrees C also co-reverted for minichromosome and CIIIR maintenance. The results suggest that ARS malfunctioning leads to minichromosome and chromosome loss from mutant cells, affecting their growth at higher temperatures.

Adenosine Triphosphatases↗

PubMed2000

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction.

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

Adenosine Triphosphatases↗

PubMed2002

Microbial transformation of elements: the case of arsenic and selenium.

Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table. These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles. Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized. Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations. Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e. the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g. FeNiSe hydrogenase). Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC). The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria. Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments. The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g. convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements.

Adenosine Triphosphatases↗

PubMed2005

Arsenate respiratory reductase gene (arrA) for Desulfosporosinus sp. strain Y5.

Desulfosporosinus sp. strain Y5 is a spore-forming bacterium capable of dissimilatory arsenate reduction coupled to the oxidation of aromatic compounds. In arsenate respiration, the arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite. Our objective is to characterize the arrA gene, encoding the ARR, for Desulfosporosinus sp. strain Y5. Oligonucleotide primers were designed based on the few arrA gene sequences available at the time and validated against positive and negative controls. The resulting arrA-amplicon of approximately 2.0kb was cloned and sequenced. The arrA from Desulfosporosinus sp. Y5 is closely related to Desulfitobacterium hafniense (similarity of 77% and 81% at the nucleotide and amino acid levels, respectively). Phylogenetic topology based on the arrA gene was partially congruent with that of 16S rRNA-based analysis. This arrA sequence will support the development of specific tracking probes for Desulfosporosinus sp. Y5 and the molecular characterization and monitoring of dissimilatory arsenate reducing bacteria.

Amino Acid Sequence↗

PubMed2006

Interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family.

In the thioredoxin (Trx)-coupled arsenate reductase family, arsenate reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled arsenate reductases. In the P-loop of the Trx-coupled arsenate reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in beta conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the pK(a) value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k(cat) value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled arsenate reductases.

Amino Acid Sequence↗

PubMed2004

Identification and quantification of arsC genes in environmental samples by using real-time PCR.

The arsC gene is responsible for the first step in arsenate biotransformation encoding the enzyme arsenate reductase. The quantitative real-time PCR method was developed to quantify the abundance of the arsC genes in environmental samples contaminated with arsenic. Two sets of primers that showed high specificity for the target arsC gene were designed based on consensus sequences from 13 bacterial species. The arsC gene was used as an external standard instead of total DNA in the calibration curve for real-time PCR, which was linear over six orders of magnitude and the detection limit was estimated to be about three copies of the gene. Soil samples from arsenic contaminated sites were screened for arsC genes by using PCR and showed the presence of this gene. The copy numbers of the gene ranging from 0.88 x 10(4) to 1.56 x 10(5) per ng total DNA were found in eight arsenic contaminated samples. Soil samples from a bioreactor containing pulp mill biomass and high concentration of arsenate showed a tenfold higher count of arsC gene copies than soil samples collected underground from an arsenic-rich gold mine.

Arsenates↗

PubMed2002

Biochemistry of arsenic detoxification.

All living organisms have systems for arsenic detoxification. The common themes are (a) uptake of As(V) in the form of arsenate by phosphate transporters, (b) uptake of As(III) in the form of arsenite by aquaglyceroporins, (c) reduction of As(V) to As(III) by arsenate reductases, and (d) extrusion or sequestration of As(III). While the overall schemes for arsenic resistance are similar in prokaryotes and eukaryotes, some of the specific proteins are the products of separate evolutionary pathways.

Amino Acid Sequence↗

PubMed2001

Enzymatic methylation of arsenic compounds. IX. Liver arsenite methyltransferase and arsenate reductase activities in primates.

Inorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are missing. Although methylation of inorganic arsenic has been considered its primary mechanism for detoxification, the results of recent investigations disagree. We have investigated 17 species of non-human primates, including great apes, New and Old World monkeys and prosimians, and have found that thirteen of them lacked hepatic arsenite methyltransferase activity in vitro. Four primate species, three from the Old World genus Macaca, and one of three animals from the New World genus Saimiri, had arsenite methyltransferase activity. That all the tissues examined were viable was demonstrated by their all having arsenate reductase activity. These data suggest that methylation of inorganic arsenic is not a detoxification mechanism for many non-human primates. Thus, alternative methods of detoxifying inorganic arsenic in mammals need to be considered and investigated. In addition, there appears to be a phylogenetic component to having arsenite methyltransferase activity, as evidenced by the result of our study of the Macaca species.

Adenosine Triphosphatases↗

PubMed2003

The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10.

The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent K(m) for arsenate of 34 microM and V(max) of 2.5 micromol min(-1) mg(-1). Optimal activity occurred at pH 9.5 and 150 g l(-1) of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins.

Arsenates↗

PubMed2003

Identification of a triad of arginine residues in the active site of the ArsC arsenate reductase of plasmid R773.

ArsC from plasmid R773 catalyzes reduction of arsenate in Escherichia coli. Arg-60, Arg-94 and Arg-107 are near the active site residue Cys-12, suggesting that they form an anion binding pocket in the active site and/or participate in catalysis. These three arginine residues were altered to a variety of other residues by site-directed mutagenesis. Only mutants with arginine-to-lysine substitutions conferred arsenate resistance in vivo, although purified R60A, R60E, R60K exhibited varying levels of enzymatic activity. The data support the hypothesis that this triad of arginine residues is involved in arsenate binding and transition-state stabilization.

Arginine↗

PubMed2000

Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli.

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.

Adenosine Triphosphatases↗

PubMed2003

Purification of an oxidation-sensitive enzyme, pI258 arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K(a)=1.1 mM) and potassium was stabilizing ArsC (DeltaT(m)=+6.2 degrees C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.

Arsenite Transporting ATPases↗

PubMed1994

Properties of the arsenate reductase of plasmid R773.

Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

Adenosine Triphosphatases↗

PubMed1994

Arsenate reductase of Staphylococcus aureus plasmid pI258.

Arsenate reductase encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 was overproduced in Escherichia coli and purified. The purified enzyme reduced radioactive arsenate to arsenite when coupled to thioredoxin, thioredoxin reductase, and NADPH. NADPH oxidation coupled to arsenate reduction also required thioredoxin and thioredoxin reductase. Glutaredoxin and reduced glutathione did not stimulate arsenate reduction. NADPH oxidation showed Michaelis-Menten kinetics with a Km of 1 microM AsO4(3-) and an apparent Vmax of 200 nmol/min per mg of protein. At high substrate concentration (above 1 mM AsO4(3-), a secondary rise in the reaction rate was observed, with a Km of 2 mM and an apparent Vmax of 450 nmol/min per mg of protein. This secondary rise also occurred upon addition of phosphate or nitrate (which were not substrates for the enzyme). Arsenite (the product of the enzyme), tellurite, and antimonite [Sb(III)] were inhibitors. Selenate (but not selenite or sulfate) was a substrate for reductase-dependent NADPH oxidation, with an apparent Km of 13 mM SeO4(2-). Arsenate reductase was purified as a monomer of 14.5 kDa, consistent with the DNA sequence. Electrospray mass spectrometry showed two molecular masses of 14,810.5 and 14,436.0 Da, suggesting that 70% of the purified protein lacked the N-terminal three amino acids; HPLC coupled to electrospray mass spectroscopy of protease digest products confirmed this conclusion and verified the entire amino acid sequence.

Adenosine Triphosphatases↗

PubMed2001

Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis.

MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.

Acyltransferases↗