Sample records for alkanesulfonates

  1. Exposing the Alkanesulfonate Monooxygenase Protein-Protein Interaction Sites. (United States)

    Dayal, Paritosh V; Singh, Harsimran; Busenlehner, Laura S; Ellis, Holly R


    The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein-protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen-deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein-protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein-protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation.

  2. The weathervane model, a functional and structural organization of the two-component alkanesulfonate oxidoreductase SsuD from Xanthomonas citri

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    Pegos, V.R. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil); Oliveira, P.S.L.; Balan, A. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil)


    Full text: In Xanthomonas citri, the phytopathogen responsible for the canker citrus disease, we identified in the ssuABCDE operon, genes encoding the alkanesulfonate ABC transporter as well as the two enzymes responsible for oxido reduction of the respective substrates. SsuD and SsuE proteins represent a two-component system that can be assigned to the group of FMNH{sub 2} -dependent monooxygenases. How- ever, despite of the biochemical information about SsuD and SsuE orthologs from Escherichia coli, there is no structural information of how the two proteins work together. In this work, we used ultracentrifugation, SAXS data and molecular modeling to construct a structural/functional model, which consists of eight molecules organized in a weathervane shape. Through this model, SsuD ligand-binding site for NADPH{sub 2} and FMN substrates is clearly exposed, in a way that might allow the protein-protein interactions with SsuE. Moreover, based on molecular dynamics simulations of SsuD in apo state, docked with NADPH{sub 2}, FMN or both substrates, we characterized the residues of the pocket, the mechanism of substrate interaction and transfer of electrons from NADPH{sub 2} to FMN. This is the first report that links functional and biochemical data with structural analyses. (author)

  3. Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization. (United States)

    Müller, Daniel B; Schubert, Olga T; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A


    Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota.

  4. Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp. (United States)

    Denger, K; Laue, H; Cook, A M


    Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.

  5. Separation of ions in acidic solution by capillary electrophoresis

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    Thornton, M.


    Capillary electrophoresis (CE) is an effective method for separating ionic species according to differences in their electrophoretic mobilities. CE separations of amino acids by direct detection are difficult due to their similar electrophoretic mobilities and low absorbances. However, native amino acids can be separated by CE as cations at a low pH by adding an alkanesulfonic acid to the electrolyte carrier which imparts selectivity to the system. Derivatization is unnecessary when direct UV detection is used at 185 nm. Simultaneous speciation of metal cations such as vanadium (IV) and vanadium (V) can easily be performed without complexation prior to analysis. An indirect UV detection scheme for acidic conditions was also developed using guanidine as the background carrier electrolyte (BCE) for the indirect detection of metal cations. Three chapters have been removed for separate processing. This report contains introductory material, references, and general conclusions. 80 refs.

  6. Experimental facility for radiation sulfoxidation of n-paraffins

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    Drozdov, A.S.; Dzhagatspanyan, R.V.; Lyaskin, Yu.G.; Soin, A.L.; Soboler, I.A.


    Sulfoxidation of n-paraffins represents practical interest as a method for direct production of alkanesulfonic acids from n-paraffins and gaseous SO/sub 2/ and O/sub 2/; alkanesulfonic acids are intermediates for synthesis of valuable biodegradable surface-active substances - sodium alkane sulfonates, which are widely used as the basis for synthetic detergents and as emulsifiers and polymerization processes. A special feature of the sulfoxidation process is the fact that the intermediate and end products, being sparingly soluble in paraffins, are separated into a separate, acid phase. On the one hand, this leads to an increase of the yield of di- and polysulfonic acids and, on the other hand, to resinification of the reaction mixture as a result of the acceleration of side processes in the acid phase. From this follows the need for rapid separation of the reaction products from the paraffin phase and prevention of side processes leading to resinification. When the sulfoxidation process is initiated photochemically, this condition is realized by the introduction of water into the reaction zone; water extracts the end products and breaks down the intermediate compound - alkanepersulfonic acids - via the reaction: H/sub 2/O + SO/sub 2/ + RSO/sub 2/O/sub 2/H ..-->.. RSO/sub 3/H + H/sub 2/SO/sub 4/. However, while decomposing the persulfonic acids, water prevents development of a degenerate-branched chain process. As a result, the reaction takes place with short unbranched chains with low quantum yield, which results in high energy costs for photochemical sulfoxidation. The radiation method of initiating the sulfoxidation reaction is attractive because of the possibility of carrying out the process in a regime of degenerate chain branching.

  7. The cys regulon of Xanthomonas citri

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    Moutran, A.; Balan, A. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil)


    Full text: In Escherichia coli, genes involved in metabolic pathway of sulfate and sulfonate compounds are clustered in a cys regulon, which includes three ABC transport system (operons: sbpcysWUA; ssuABC and tauABC), thirteen genes involved in the sulfur reduction (ssuDE; tauD and cysDNCHIJGK) and two regulatory proteins that belong to LysR transcription family: CysB and Cbl. Notably, a search and comparative analysis of these genes in the genomes of the citrus pathogen Xanthomonas citri and other phylogenetically related Xanthomonas species revealed the presence of genes involved with alkanesulfonate, sulfate ester and taurine, only in X. citri, suggesting that proteins from this regulon might be associated with pathogenicity in citrus. Using the molecular modeling associated with a system biology view, we modeled all the protein structures of the X. citri cys regulon as well as characterized the important residues forming the putative active sites. Comparison with orthologs from different microorganisms was made in order to get a phylogenetic relationships. We showed that proteins that are responsible for the affinity and specificity of the alkanesulfonate, sulfate and taurine transport systems conserved the residues involved in the sulfate coordination but are organized in different branches in evolution. Inside these phylogenetic branches, proteins involved in the sulfate transporter are highly conserved when compared to the others. Moreover, we identified that the taurine-binding protein (TauA) of the X. citri belongs to a different evolutionary branch from that one that described for E. coli. These differences were also noticed for components of the tau operon, including a putative new regulator. The function and mechanism of action of each protein is discussed in order to bring light for the sulfur assimilation processes and their importance for X. citri physiology. (author)

  8. A novel, fast responding, low noise potentiometric sensor containing a carbon-based polymeric membrane for measuring surfactants in industrial and environmental applications. (United States)

    Samardžić, Mirela; Galović, Olivera; Hajduković, Mateja; Sak-Bosnar, Milan


    A new high-sensitivity potentiometric sensor for anionic surfactants was fabricated using the dimethyldioctadecylammonium-tetraphenylborate (DDA-TPB) ion associate as an ionophore that was incorporated into a liquid PVC membrane. Carbon powder was used for immobilization of the ionophore in the membrane, thus significantly reducing its ohmic resistance and reducing its signal drift. The sensor exhibits a sub-Nernstian response for both dodecylbenzenesulfonate (DBS) and dodecyl sulfate (DS) in H2O (55.3 and 58.5mV/decade of activity, respectively) in a range between 3.2×10(-7) and 4.6×10(-3)M for DS and 2.5×10(-7) and 1.2×10(-3)M for DBS. The sensor also exhibited a sub-Nernstian response for DS and DBS in 10mM Na2SO4 (55.4 and 57.7mV/decade of activity, respectively) between 2.5×10(-7) and 4.6×10(-3)M for DS and 1.5×10(-7) and 8.8×10(-4)M for DBS. The detection limits for DS and DBS in H2O were 2.5×10(-7) and 2.0×10(-7) M and in 10mM Na2SO4 the detection limits were 2.5×10(-7) and 1.2×10(-7) M, respectively. The response time of the sensor was less than 5s for changes at higher concentration levels (above 1×10(-4)M) in both water and 10mM Na2SO4. At lower concentrations (below 1×10(-5)M) the response times were 8 and 6s in water and 10mM Na2SO4, respectively. The signal drift of the sensor was 1.2mV/hour. The new carbon-based sensor exhibited excellent selectivity performance for DS over almost all of the anions commonly present in commercial formulations and it was successfully employed as an end-point detector in potentiometric titrations of anionic surfactants in a pH range from 3 to 12. Three-component mixtures containing sodium alkanesulfonate (C10, C12 and C14) were successfully differentially titrated.

  9. Redox-Induced Backbiting of Surface-Tethered Alkylsulfonate Amphiphiles: Reversible Switching of Surface Wettability and Adherence. (United States)

    Dos Ramos, Lionel; de Beer, Sissi; Hempenius, Mark A; Vancso, G Julius


    The synthesis and characterization of electrode-supported poly(ferrocenylsilane) (PFS) films bearing iodopropyl (PFS-I) and undecanesulfonate (PFS-SO3(-)) surface moieties are presented. The redox responsiveness of these PFS films allows for controlled and repeatable switching of the surface energy of the PFS-I and PFS-SO3(-) layers under electrochemical control. Static water/surface contact angle measurements showed a change in contact angle values for PFS-I from 80° (reduced state) to 70° (oxidized state) over repeated cycles. However, an opposite change in wettability was observed for PFS-SO3(-), where the values observed varied from 59° (reduced state) to 77° (oxidized state). Nanoscale adherence was assessed with colloid probe AFM. The adhesive forces between these surfaces and a polystyrene (PS) colloid probe in water alternated between 130 nN (reduced state) and 30 nN (oxidized state) for PFS-I layers and between 75 nN (reduced) and 180 nN (oxidized) for the PFS-SO3(-) films. The reversed response of PFS-I films to oxidation compared to that of PFS-SO3(-), in both contact angles and adhesive forces, suggests a different underlying mechanism for switching. As PFS-I is tuned from the reduced to the oxidized state, positively charged ferrocenium (Fc(+)) centers that formed in the film increase its wettability and reduce its adherence to the hydrophobic colloid probe. For PFS-SO3(-) in the reduced state, the exposed alkanesulfonate moieties increase the hydrophilicity of the surface. When oxidized, the Fc(+) units attract the negatively charged sulfonate groups, which results in a bending of the sulfonate groups toward the PFS surface, exposing the undecyl spacer. This alteration of the surface chemistry reduces the surface energy and increases the adherence between the bent alkyl chains and the hydrophobic PS colloid in water. The attraction of the charged sulfonate group to Fc(+) is in competition with the counterions present in the electrolyte solution

  10. Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU. (United States)

    Laue, H; Denger, K; Cook, A M


    Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was