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Sample records for haloalkane dehalogenase linb

  1. Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry▿

    OpenAIRE

    Monincová, Marta; Prokop, Zbyněk; Vévodová, Jitka; Nagata, Yuji; Damborský, Jiří

    2007-01-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of Li...

  2. Weak activity of haloalkane dehalogenase LinB with 1,2,3-trichloropropane revealed by X-Ray crystallography and microcalorimetry.

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbynek; Vévodová, Jitka; Nagata, Yuji; Damborsky, Jirí

    2007-03-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.

  3. Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry▿

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbyněk; Vévodová, Jitka; Nagata, Yuji; Damborský, Jiří

    2007-01-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities. PMID:17259360

  4. Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB.

    Science.gov (United States)

    Gross, Johannes; Prokop, Zbyněk; Janssen, Dick; Faber, Kurt; Hall, Mélanie

    2016-08-03

    The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations

    Czech Academy of Sciences Publication Activity Database

    Hladílková, Jana; Prokop, Z.; Chaloupková, R.; Damborský, J.; Jungwirth, Pavel

    2013-01-01

    Roč. 117, č. 46 (2013), s. 14329-14335 ISSN 1520-6106 R&D Projects: GA ČR GBP208/12/G016 Grant - others:GA ČR(CZ) GAP207/12/0775 Program:GA Institutional support: RVO:61388963 Keywords : access tunnel * buried active site * catalytic activity * enzyme mechanism * haloalkane dehalogenase * halide ions Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

  6. Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

    Science.gov (United States)

    Dravis, B C; Swanson, P E; Russell, A J

    2001-11-20

    Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization. Copyright 2001 John Wiley & Sons, Inc.

  7. Stepwise dissection and visualization of the catalytic mechanism of haloalkane dehalogenase LinB using molecular dynamics simulations and computer graphics.

    Science.gov (United States)

    Negri, Ana; Marco, Esther; Damborsky, Jiri; Gago, Federico

    2007-10-01

    The different steps of the dehalogenation reaction carried out by LinB on three different substrates have been characterized using a combination of quantum mechanical calculations and molecular dynamics simulations. This has allowed us to obtain information in atomic detail about each step of the reaction mechanism, that is, substrate entrance and achievement of the near-attack conformation, transition state stabilization within the active site, halide stabilization, water molecule activation and subsequent hydrolytic attack on the ester intermediate with formation of alcohol, and finally product release. Importantly, no bias or external forces were applied during the whole procedure so that both intermediates and products were completely free to sample configuration space in order to adapt to the plasticity of the active site and/or search for an exit. Differences in substrate reactivity were found to be correlated with the ease of adopting the near-attack conformation and two different exit pathways were found for product release that do not interfere with substrate entrance. Additional support for the different entry and exit pathways was independently obtained from an examination of the enzyme's normal modes.

  8. Metagenome-derived haloalkane dehalogenases with novel catalytic properties

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Vaňáček, P.; Kuňka, A.; Prokop, Z.; Dambrovský, J.

    2017-01-01

    Roč. 101, č. 16 (2017), s. 6385-6397 ISSN 0175-7598 R&D Projects: GA ČR GAP504/10/0137; GA MŠk(CZ) LM2015047; GA MŠk(CZ) LM2015055 Institutional support: RVO:61388971 Keywords : Haloalkane dehalogenase * Metagenomic DNA * Heterologous production Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.420, year: 2016

  9. Kinetics of halide release of haloalkane dehalogenase : Evidence for a slow conformational change

    NARCIS (Netherlands)

    Schanstra, JP; Janssen, DB; Schanstra, Joost P.

    1996-01-01

    Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides, The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the

  10. REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCES HALIDE BINDING AND CATALYTIC ACTIVITY

    NARCIS (Netherlands)

    KENNES, C; PRIES, F; KROOSHOF, GH; BOKMA, E; Kingma, Jacob; JANSSEN, DB

    1995-01-01

    Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues

  11. Repositioning the catalytic triad aspartic acid of haloalkane dehalogenase : Effects on stability, kinetics, and structure

    NARCIS (Netherlands)

    Krooshof, Geja H.; Kwant, Edwin M.; Damborský, Jiří; Koča, Jaroslav; Janssen, Dick B.

    1997-01-01

    Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member

  12. Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases : From Chemical Waste to Enantiopure Building Blocks

    NARCIS (Netherlands)

    van Leeuwen, Jan G. E.; Wijma, Hein J.; Floor, Robert J.; van der Laan, Jan-Metske; Janssen, Dick B.

    2012-01-01

    We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active

  13. A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

    DEFF Research Database (Denmark)

    Næsted, Henrik; Fennema, M.; Hao, L.

    1999-01-01

    , including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhl...

  14. Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a Rhodococcus haloalkane dehalogenase

    NARCIS (Netherlands)

    Bosma, T; Pikkemaat, MG; Kingma, Jacob; Dijk, J; Janssen, DB

    2003-01-01

    Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the

  15. Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations

    Czech Academy of Sciences Publication Activity Database

    Klvana, M.; Pavlová, M.; Koudeláková, T.; Chaloupková, R.; Dvořák, P.; Prokop, Z.; Stsiapanava, A.; Kutý, Michal; Kutá-Smatanová, Ivana; Dohnálek, Jan; Kulhánek, P.; Damborský, J.

    2009-01-01

    Roč. 392, č. 5 (2009), s. 1339-1356 ISSN 0022-2836 R&D Projects: GA MŠk(CZ) LC06010 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z60870520 Keywords : haloalkane dehalogenase * product release * random acceleration molecular dynamics Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.871, year: 2009

  16. Activation of an Asp-124→Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine

    NARCIS (Netherlands)

    Pries, Frens; Kingma, Jacob; JANSSEN, Dick B

    1995-01-01

    Haloalkane dehalogenase hydrolyses various 1-halon-alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl-enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp-124) that displaces the halogen during formation of the intermediate was changed

  17. Kinetic resolution of α-bromoamides: Experimental and theoretical investigation of highly enantioselective reactions catalyzed by haloalkane dehalogenases

    NARCIS (Netherlands)

    Westerbeek, Alja; Szymanski, Wiktor; Wijma, Hein J.; Marrink, Siewert; Feringa, Ben L.; Janssen, Dick B.

    2011-01-01

    Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic alpha-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate

  18. Directed evolution strategies for enantiocomplementary haloalkane dehalogenases: from chemical waste to enantiopure building blocks.

    Science.gov (United States)

    van Leeuwen, Jan G E; Wijma, Hein J; Floor, Robert J; van der Laan, Jan-Metske; Janssen, Dick B

    2012-01-02

    We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene

    OpenAIRE

    Bosma, Tjibbe; Damborský, Jiří; Stucki, Gerhard; Janssen, Dick B.

    2002-01-01

    Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, p...

  20. Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

    International Nuclear Information System (INIS)

    Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

    2012-01-01

    The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution. Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit

  1. Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene.

    Science.gov (United States)

    Bosma, Tjibbe; Damborský, Jirí; Stucki, Gerhard; Janssen, Dick B

    2002-07-01

    Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.

  2. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Directory of Open Access Journals (Sweden)

    Anzhang Li

    Full Text Available Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m = 0.82 mM, k(cat/K(m = 16.43 mM(-1 · s(-1. DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  3. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Science.gov (United States)

    Li, Anzhang; Shao, Zongze

    2014-01-01

    Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m) = 0.82 mM, k(cat)/K(m) = 16.43 mM(-1) · s(-1)). DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  4. Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31.

    Science.gov (United States)

    Lahoda, Maryna; Mesters, Jeroen R; Stsiapanava, Alena; Chaloupkova, Radka; Kuty, Michal; Damborsky, Jiri; Kuta Smatanova, Ivana

    2014-02-01

    Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

  5. Conformational Changes Allow Processing of Bulky Substrates by a Haloalkane Dehalogenase with a Small and Buried Active Site.

    Science.gov (United States)

    Kokkonen, Piia; Bednar, David; Dockalova, Veronika; Prokop, Zbynek; Damborsky, Jiri

    2018-06-01

    Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and which is likely the route for bulky substrates. These results provide insights into the structure-dynamics-function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to DCE introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling.

    Science.gov (United States)

    Marques, Sérgio M; Dunajova, Zuzana; Prokop, Zbynek; Chaloupkova, Radka; Brezovsky, Jan; Damborsky, Jiri

    2017-08-28

    The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the S N 2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust

  7. Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling

    Science.gov (United States)

    Banáš, Pavel; Otyepka, Michal; Jeřábek, Petr; Petřek, Martin; Damborský, Jiří

    2006-06-01

    1,2,3-Trichloropropane (TCP) is a highly toxic, recalcitrant byproduct of epichlorohydrin manufacture. Haloalkane dehalogenase (DhaA) from Rhodococcus sp. hydrolyses the carbon-halogen bond in various halogenated compounds including TCP, but with low efficiency ( k cat/ K m = 36 s-1 M-1). A Cys176Tyr-DhaA mutant with a threefold higher catalytic efficiency for TCP dehalogenation has been previously obtained by error-prone PCR. We have used molecular simulations and quantum mechanical calculations to elucidate the molecular mechanisms involved in the improved catalysis of the mutant, and enantioselectivity of DhaA toward TCP. The Cys176Tyr mutation modifies the protein access and export routes. Substitution of the Cys residue by the bulkier Tyr narrows the upper tunnel, making the second tunnel "slot" the preferred route. TCP can adopt two major orientations in the DhaA enzyme, in one of which the halide-stabilizing residue Asn41 forms a hydrogen bond with the terminal halogen atom of the TCP molecule, while in the other it bonds with the central halogen atom. The differences in these binding patterns explain the preferential formation of the ( R)- over the ( S)-enantiomer of 2,3-dichloropropane-1-ol in the reaction catalyzed by the enzyme.

  8. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    Science.gov (United States)

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations.

    Science.gov (United States)

    Klvana, Martin; Pavlova, Martina; Koudelakova, Tana; Chaloupkova, Radka; Dvorak, Pavel; Prokop, Zbynek; Stsiapanava, Alena; Kuty, Michal; Kuta-Smatanova, Ivana; Dohnalek, Jan; Kulhanek, Petr; Wade, Rebecca C; Damborsky, Jiri

    2009-10-09

    Eight mutants of the DhaA haloalkane dehalogenase carrying mutations at the residues lining two tunnels, previously observed by protein X-ray crystallography, were constructed and biochemically characterized. The mutants showed distinct catalytic efficiencies with the halogenated substrate 1,2,3-trichloropropane. Release pathways for the two dehalogenation products, 2,3-dichloropropane-1-ol and the chloride ion, and exchange pathways for water molecules, were studied using classical and random acceleration molecular dynamics simulations. Five different pathways, denoted p1, p2a, p2b, p2c, and p3, were identified. The individual pathways showed differing selectivity for the products: the chloride ion releases solely through p1, whereas the alcohol releases through all five pathways. Water molecules play a crucial role for release of both products by breakage of their hydrogen-bonding interactions with the active-site residues and shielding the charged chloride ion during its passage through a hydrophobic tunnel. Exchange of the chloride ions, the alcohol product, and the waters between the buried active site and the bulk solvent can be realized by three different mechanisms: (i) passage through a permanent tunnel, (ii) passage through a transient tunnel, and (iii) migration through a protein matrix. We demonstrate that the accessibility of the pathways and the mechanisms of ligand exchange were modified by mutations. Insertion of bulky aromatic residues in the tunnel corresponding to pathway p1 leads to reduced accessibility to the ligands and a change in mechanism of opening from permanent to transient. We propose that engineering the accessibility of tunnels and the mechanisms of ligand exchange is a powerful strategy for modification of the functional properties of enzymes with buried active sites.

  10. Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I

    International Nuclear Information System (INIS)

    Bogdanović, Xenia; Hesseler, Martin; Palm, Gottfried J.; Bornscheuer, Uwe T.; Hinrichs, Winfried

    2010-01-01

    The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported. DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2 1 2 1 2. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator

  11. Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature.

    Science.gov (United States)

    Chaloupkova, Radka; Prokop, Zbynek; Sato, Yukari; Nagata, Yuji; Damborsky, Jiri

    2011-08-01

    The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase. © 2011 The Authors Journal compilation © 2011 FEBS.

  12. Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels.

    Science.gov (United States)

    Stsiapanava, A; Dohnalek, J; Gavira, J A; Kuty, M; Koudelakova, T; Damborsky, J; Kuta Smatanova, I

    2010-09-01

    The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

  13. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  14. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  15. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

    Czech Academy of Sciences Publication Activity Database

    Stsiapanava, A.; Chaloupková, R.; Fořtová, A.; Brynda, Jiří; Weiss, M.S.; Damborský, J.; Kutá-Smatanová, Ivana

    2011-01-01

    Roč. 67, - (2011), s. 253-257 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010 Grant - others:GA ČR(CZ) GA310/09/1407 Program:GA Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z60870520 Keywords : haloalkane dehalogenases * DhaA * Rhodococcus rhodochrous * microseeding * atomic resolution Subject RIV: EB - Genetics ; Molecular Biology; CD - Macromolecular Chemistry (UEK-B) Impact factor: 0.506, year: 2011

  16. Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism

    NARCIS (Netherlands)

    Poelarends, GJ; Bosma, T; Kulakov, LA; Larkin, MJ; Marchesi, [No Value; Weightman, AJ; Janssen, DB; Kulakov, Leonid A.; Larkin, Michael J.; Marchesi, Julian R.; Weightman, Andrew J.

    The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared.

  17. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

    International Nuclear Information System (INIS)

    Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S.; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-01-01

    Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution. Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2 1 2 1 2 1 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively

  18. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands.

    Science.gov (United States)

    Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta

    2011-02-01

    Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

  19. Metabolomics of hexachlorocyclohexane (HCH) transformation: ratio of LinA to LinB determines metabolic fate of HCH isomers.

    Science.gov (United States)

    Geueke, Birgit; Garg, Nidhi; Ghosh, Sneha; Fleischmann, Thomas; Holliger, Christof; Lal, Rup; Kohler, Hans-Peter E

    2013-04-01

    Although the production and use of technical hexachlorocyclohexane (HCH) and lindane (the purified insecticidal isomer γ-HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ-HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α-, β-, γ-, δ-, ε-HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ-HCH degradation, a dehydrochlorinase (LinA) and a haloalkane dehalogenase (LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ-HCH and that under these conditions other HCH-isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side-products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  20. Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

    Science.gov (United States)

    Jesenská, Andrea; Sedlác̆ek, Ivo; Damborský, Jir̆í

    2000-01-01

    Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment. PMID:10618227

  1. Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene

    DEFF Research Database (Denmark)

    van der Ploeg, J; Van Hall, Gerrit; Janssen, D B

    1991-01-01

    B) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology...... chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhl...... (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10....

  2. Comparison of formation of reactive conformers for the SN2 displacements by CH3CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} in water and by Asp124-CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} in a haloalkane dehalogenase

    Science.gov (United States)

    Hur, Sun; Kahn, Kalju; Bruice, Thomas C.

    2003-01-01

    The SN2 displacement of Cl− from 1,2-dichloroethane by acetate (CH3CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document}) in water and by the carboxylate of the active site aspartate in the haloalkane dehalogenase of Xanthobacter autothropicus have been compared by using molecular dynamics simulations. In aqueous solution, six families of contact-pair structures (I–VI) were identified, and their relative concentrations and dissociation rate constants were determined. The near attack conformers (NACs) required for the SN2 displacement reaction are members of the IV (CH3COO−⋅ ⋅ ⋅CH2(Cl)CH2Cl) family and are formed in the sequence II→III→IV→NAC. The NAC subclass is defined by the —COO−⋅ ⋅ ⋅C—Cl contact distance of ≤3.41 Å and the —COO−⋅ ⋅ ⋅C—Cl angle of 157–180°. The mole percentage of NACs is 0.16%, based on the 1 M standard state. This result may be compared with 13.4 mole percentage of NACs in the Michaelis complex in the enzyme. It follows that NAC formation in the enzyme is favored by 2.6 kcal/mol. Because reaction coordinates from S to TS, both in water and in the enzyme, pass via NAC (i.e., S → NAC → TS), the reduction in the S → NAC barrier by 2.6 kcal/mol accounts for ≈25% of the reduction of total barrier in the S → TS (10.7 kcal/mol). The remaining 75% of the advantage of the enzymatic reaction revolves around the efficiency of NAC → TS step. This process, based on previous studies, is discussed briefly. PMID:12610210

  3. Sequence diversity in haloalkane dehalogenases, as revealed by PCR using family-specific primers

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Faměrová, Veronika

    2012-01-01

    Roč. 88, č. 2 (2012), s. 212-217 ISSN 0167-7012 R&D Projects: GA ČR GAP504/10/0137; GA ČR GAP207/10/0135 Institutional research plan: CEZ:AV0Z50200510 Keywords : Dehalogenation * Consensus sequence * Degenerate PCR primer Subject RIV: EE - Microbiology, Virology Impact factor: 2.161, year: 2012

  4. Nanosecond Time-Dependent Stokes Shift at the Tunnel Mouth of Haloalkane Dehalogenases

    Czech Academy of Sciences Publication Activity Database

    Jesenská, A.; Sýkora, Jan; Olžyńska, Agnieszka; Brezovský, J.; Zdráhal, Z.; Damborský, J.; Hof, Martin

    2009-01-01

    Roč. 131, č. 2 (2009), s. 494-501 ISSN 0002-7863 R&D Projects: GA ČR GA203/08/0114; GA MŠk(CZ) LC06063; GA MŠk(CZ) LC06010 Institutional research plan: CEZ:AV0Z40400503 Keywords : crystallographic analysis * active-site dynamics * solvent relaxation Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 8.580, year: 2009

  5. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate.

    Science.gov (United States)

    Pavlova, Martina; Klvana, Martin; Prokop, Zbynek; Chaloupkova, Radka; Banas, Pavel; Otyepka, Michal; Wade, Rebecca C; Tsuda, Masataka; Nagata, Yuji; Damborsky, Jiri

    2009-10-01

    Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

  6. Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31

    Czech Academy of Sciences Publication Activity Database

    Lahoda, M.; Mesters, J. R.; Stsiapanava, A.; Chaloupková, R.; Kutý, Michal; Damborský, J.; Kutá-Smatanová, Ivana

    2014-01-01

    Roč. 70, FEB 2014 (2014), s. 209-217 ISSN 0907-4449 Institutional support: RVO:67179843 Keywords : DhaA31 * substrate-free * 3rk4 * complex with TCP * 4fwb * wild-type DhaA * 4hzg Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  7. Differences in crystallization of two LinB variants from Sphingobium japonicum UT26

    Czech Academy of Sciences Publication Activity Database

    Degtjarik, O.; Chaloupková, R.; Řezáčová, Pavlína; Kutý, M.; Damborský, J.; Smatanová, I.K.

    2013-01-01

    Roč. 69, č. 3 (2013), s. 284-287 ISSN 1744-3091 Institutional support: RVO:68378050 Keywords : protein crystallization * dehalogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.568, year: 2013

  8. A review on non-stereospecific haloalkanoic acid dehalogenases ...

    African Journals Online (AJOL)

    Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,LDEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D- and L- ...

  9. Influence of Li/Nb ratios on defect structure and photorefractive properties of Zn: In: Fe: LiNbO 3 crystals

    Science.gov (United States)

    Dai, Li; Su, Yan-Qing; Wu, Shi-Ping; Guo, Jing-Jie; Xu, Chao; Xu, Yu-Heng

    2011-04-01

    A series of Zn: In: Fe: LiNbO3 crystals are grown by the Czochralski technique with various ratios of Li/Nb = 0.94, 1.05, 1.20 and 1.38 in the melt. The Zn, In, Fe, Nb and Li concentrations in the crystals are analyzed by inductively coupled plasma (ICP) spectrometry. The results indicate that with increasing the [Li]/[Nb] ratio in melt, [Li]/[Nb] ratio increases and goes up continuously in the crystal, the segregation coefficients of both Zn and In ions decrease. The absorption spectra measurement and two-wave coupling experiment are employed to study the effect of [Li]/[Nb] ratio on photorefractive properties of Zn: In: Fe: LiNbO3 crystals. It is found that the [Li]/[Nb] ratio increases, the write time is shortened and the photorefractive sensitivity is improved.

  10. Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Tang, Lixia; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB

    2003-01-01

    Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic

  11. Chloromuconolactone dehalogenase ClcF of actinobacteria.

    Science.gov (United States)

    Solyanikova, Inna P; Plotnikova, Elena G; Shumkova, Ekaterina S; Robota, Irina V; Prisyazhnaya, Natalya V; Golovleva, Ludmila A

    2014-01-01

    This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram-positive bacteria, Rhodococcus opacus 1CP, adapted to 2-chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R. opacus 1CP. CMLDs isolated from the biomass of strains Rhodococcus spp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of the clcF gene from the ClcF of strain R. opacus 1CP. Matrix-assisted laser dissociation/ionization time-of-flight mass spectrometry showed the presence of a peak with m/z 11,194-11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats.

  12. Effect of [Li]/[Nb] ratio on composition and defect structure of Zr:Yb:Tm:LiNbO3 crystals

    Science.gov (United States)

    Liu, Chunrui; Dai, Li; Wang, Luping; Shao, Yu; Yan, Zhehua; Xu, Yuheng

    2018-04-01

    Zr:Yb:Tm:LiNbO3 crystals with various [Li]/[Nb] ratios (0.946, 1.05, 1.20 and 1.38) were grown by the Czochralski technique. Distribution coefficients of Zr4+, Yb3+ and Tm3+ ions were analyzed by the inductively coupled plasma-atomic emission spectrometer (ICP-AES). The influence of [Li]/[Nb] ratio on the composition and defect structure of Zr:Yb:Tm:LiNbO3 crystals was investigated by X-ray diffraction and IR transmission spectrum. The results show that as the [Li]/[Nb] ratio increases in the melt, the distribution coefficients of Yb3+ and Tm3+ ions both increase while that of Zr4+ ion deceases. When the [Li]/[Nb] ratio increases to 1.20 in the melt, Zr:Yb:Tm:LiNbO3 crystal is nearly stoichiometric. In addition, when the [Li]/[Nb] ratio reaches up to 1.38, NbLi4+ are completely replaced and Li+ starts to impel the Zr4+, Yb3+ and Tm3+ into the normal Li sites.

  13. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    Jong, René M. de; Rozeboom, Henriëtte J.; Kalk, Kor H.; Tang, Lixia; Janssen, Dick B.; Dijkstra, Bauke W.

    2002-01-01

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  14. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    de Jong, RM; Rozeboom, HJ; Kalk, KH; Tang, Lixia; Janssen, DB; Dijkstra, BW

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  15. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes : Prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    NARCIS (Netherlands)

    Jiménez Avella, Diego; Dini Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered

  16. Are Time-Dependent Fluorescence Shifts at the Tunnel Mouth of Haloalkane Dehalogenase Enzymes Dependent on the Choice of the Chromophore?

    Czech Academy of Sciences Publication Activity Database

    Amaro, Mariana; Brezovský, J.; Kováčová, S.; Maier, L.; Chaloupková, R.; Sýkora, Jan; Paruch, K.; Damborský, J.; Hof, Martin

    2013-01-01

    Roč. 117, č. 26 (2013), s. 7898-7906 ISSN 1520-6106 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : DYNAMIC STOKES SHIFT * WATER-PROTEIN FLUCTUATIONS * POLAR SOLVATION DYNAMICS Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

  17. Role of Dehalogenases in Aerobic Bacterial Degradation of Chlorinated Aromatic Compounds

    Directory of Open Access Journals (Sweden)

    Pankaj Kumar Arora

    2014-01-01

    Full Text Available This review was conducted to provide an overview of dehalogenases involved in aerobic biodegradation of chlorinated aromatic compounds. Additionally, biochemical and molecular characterization of hydrolytic, reductive, and oxygenolytic dehalogenases was reviewed. This review will increase our understanding of the process of dehalogenation of chlorinated aromatic compounds.

  18. Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications

    Directory of Open Access Journals (Sweden)

    Thiau-Fu Ang

    2018-05-01

    Full Text Available The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

  19. Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications.

    Science.gov (United States)

    Ang, Thiau-Fu; Maiangwa, Jonathan; Salleh, Abu Bakar; Normi, Yahaya M; Leow, Thean Chor

    2018-05-07

    The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

  20. XPS study of Li/Nb ratio in LiNbO{sub 3} crystals. Effect of polarity and mechanical processing on LiNbO{sub 3} surface chemical composition

    Energy Technology Data Exchange (ETDEWEB)

    Skryleva, E.A., E-mail: easkryleva@gmail.com; Kubasov, I.V., E-mail: kubasov.ilya@gmail.com; Kiryukhantsev-Korneev, Ph.V., E-mail: kiruhancev-korneev@yandex.ru; Senatulin, B.R., E-mail: borisrs@yandex.ru; Zhukov, R.N., E-mail: rom_zhuk@mail.ru; Zakutailov, K.V., E-mail: zakkonst@gmail.com; Malinkovich, M.D., E-mail: malinkovich@yandex.ru; Parkhomenko, Yu.N., E-mail: parkh@rambler.ru

    2016-12-15

    Highlights: • XPS Li/Nb ratio measurement uncertainty in LNbO3 specimens was obtained. • The effect of polarization on surface chemistry was observed only on cleaves. • Li/Nb ratio on positive cleave surface is higher than on negative one. • The positive cleave surface adsorbs fluorine more efficiently than negative one. • Mechanical processing of crystals reduces surface Li/Nb. - Abstract: Different sections of congruent lithium niobate (CLN) crystals have been studied using X-ray photoelectron spectroscopy (XPS). We have developed a method for measuring the lithium-to-niobium atomic ratio Li/Nb from the ratio of the Li1s and Nb4s spectral integral intensities with an overall error of within 8 %. Polarity and mechanical processing affect the Li/Nb ratio on CLN crystal surfaces. The Li/Nb ratio is within the tolerance (0.946 ± 0.074) on the negative cleave surface Z, and there is excess lithium (Li/Nb = 1.25 ± 0.10) on the positive surface. The positive surfaces of the 128° Y cut plates after long exposure to air exhibit LiOH formation indications (obvious lithium excess, higher Li1s spectral binding energy and a wide additional peak in the O1s spectrum produced by nonstructural oxygen). XPS and glow discharge optical electron spectroscopy showed that mechanical processing of differently oriented crystals (X, Z and 128° Y) and different polarities dramatically reduces the Li/Nb ratio. In situ fluorine adsorption experiments revealed the following regularities: fluorine adsorption only occurred on crystal cleaves and was not observed for mechanically processed specimens. Positive cleave surfaces have substantially higher fluorine adsorption capacity compared to negative ones.

  1. Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

    Science.gov (United States)

    Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

    2014-12-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense

    DEFF Research Database (Denmark)

    Christensen, Nina; Ahring, Birgitte Kiær; Wohlfarth, Gert

    1998-01-01

    The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol- educing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive...... dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS-PAGErevealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68±0.2 mol corrinoid, 12.0±0.7 mol iron, and 13...

  3. Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

    NARCIS (Netherlands)

    Maillard, J.; Schumacher, W.; Vazquez, F.; Regeard, C.; Hagen, W.R.; Holliger, C.

    2003-01-01

    The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a

  4. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

    Science.gov (United States)

    Payne, Karl Ap; Quezada, Carolina P; Fisher, Karl; Dunstan, Mark S; Collins, Fraser A; Sjuts, Hanno; Levy, Colin; Hay, Sam; Rigby, Stephen Ej; Leys, David

    2015-01-22

    Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

  5. Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by L-2-Haloacid Dehalogenase

    NARCIS (Netherlands)

    Ridder, Ivo S.; Rozeboom, Henriëtte J.; Kalk, Kor H.; Dijkstra, Bauke W.

    1999-01-01

    The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C2 atom. The structure of the

  6. Diversity of reductive dehalogenase genes from environmental samples and enrichment cultures identified with degenerate primer PCR screens.

    Directory of Open Access Journals (Sweden)

    Laura Audrey Hug

    2013-11-01

    Full Text Available Reductive dehalogenases are the critical enzymes for anaerobic organohalide respiration, a microbial metabolic process that has been harnessed for bioremediation efforts to resolve chlorinated solvent contamination in groundwater and is implicated in the global halogen cycle. Reductive dehalogenase sequence diversity is informative for the dechlorination potential of the site or enrichment culture. A suite of degenerate PCR primers targeting a comprehensive curated set of reductive dehalogenase genes was designed and applied to twelve DNA samples extracted from contaminated and pristine sites, as well as six enrichment cultures capable of reducing chlorinated compounds to non-toxic end-products. The amplified gene products from four environmental sites and two enrichment cultures were sequenced using Illumina HiSeq, and the reductive dehalogenase complement of each sample determined. The results indicate that the diversity of the reductive dehalogenase gene family is much deeper than is currently accounted for: one-third of the translated proteins have less than 70% pairwise amino acid identity to database sequences. Approximately 60% of the sequenced reductive dehalogenase genes were broadly distributed, being identified in four or more samples, and often in previously sequenced genomes as well. In contrast, 17% of the sequenced reductive dehalogenases were unique, present in only a single sample and bearing less than 90% pairwise amino acid identity to any previously identified proteins. Many of the broadly distributed reductive dehalogenases are uncharacterized in terms of their substrate specificity, making these intriguing targets for further biochemical experimentation. Finally, comparison of samples from a contaminated site and an enrichment culture derived from the same site eight years prior allowed examination of the effect of the enrichment process.

  7. Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures.

    Science.gov (United States)

    Wang, Yayue; Feng, Yanbin; Cao, Xupeng; Liu, Yinghui; Xue, Song

    2018-01-23

    D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H 2 18 O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats. The product-bound complex structure was trapped with L-lactic acid in the active site, which is framed by the structurally related helices between two repeats. Site-directed mutagenesis confirmed the importance of the residues lining the binding pocket in stabilizing the enzyme-substrate complex. Asp205 acts as a key catalytic residue and is responsible for activating a water molecule along with Asn131. Then, the hydroxyl group of the water molecule directly attacks the C2 atom of the substrate to release the halogen ion instead of forming an enzyme-substrate ester intermediate as observed in L-2-haloacid dehalogenases. The newly revealed structural and mechanistic information on D-DEX may inspire structure-based mutagenesis to engineer highly efficient haloacid dehalogenases.

  8. Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States); Serrano, Hector; Whitman, Christian P. [Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin (United States); Mesecar, Andrew D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States)

    2008-12-01

    The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed. Trans-3-chloroacrylic acid dehalogenase (CaaD) is a critical enzyme in the trans-1, 3-dichloropropene (DCP) degradation pathway in Pseudomonas pavonaceae 170. This enzyme allows bacteria to use trans-DCP, a common component in commercially produced fumigants, as a carbon source. CaaD specifically catalyzes the fourth step of the pathway by cofactor-independent dehalogenation of a vinyl carbon–halogen bond. Previous studies have reported an X-ray structure of CaaD under acidic conditions with a covalent modification of the catalytic βPro1 residue. Here, the 1.7 Å resolution X-ray structure of CaaD under neutral (pH 6.5) conditions is reported without the presence of the covalent adduct. In this new structure, a substrate-like acetate molecule is bound within the active site in a position analogous to the putative substrate-binding site. Additionally, a catalytically important water molecule was identified, consistent with previously proposed reaction schemes. Finally, flexibility of the catalytically relevant side chain αGlu52 is observed in the structure, supporting its role in the catalytic mechanism.

  9. Dehalogenation Activities and Distribution of Reductive Dehalogenase Homologous Genes in Marine Subsurface Sediments▿ †

    Science.gov (United States)

    Futagami, Taiki; Morono, Yuki; Terada, Takeshi; Kaksonen, Anna H.; Inagaki, Fumio

    2009-01-01

    Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem. PMID:19749069

  10. Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation

    Directory of Open Access Journals (Sweden)

    Bat-Erdene eJugder

    2016-03-01

    Full Text Available Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e. organohalide respiration. Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDase, which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses RDases, concentrating on those which have been purified (partially or wholly and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

  11. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2016-01-01

    Full Text Available 2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA, conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K and aspartate (D residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models.

  12. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane

    KAUST Repository

    Merlino, Giuseppe

    2015-03-06

    The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales . Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community.

  13. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane

    KAUST Repository

    Merlino, Giuseppe; Balloi, Annalisa; Marzorati, Massimo; Mapelli, Francesca; Rizzi, Aurora; Lavazza, Davide; de Ferra, Francesca; Carpani, Giovanna; Daffonchio, Daniele

    2015-01-01

    The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales . Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community.

  14. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon.

    Science.gov (United States)

    Mattes, Timothy E; Ewald, Jessica M; Liang, Yi; Martinez, Andres; Awad, Andrew; Richards, Patrick; Hornbuckle, Keri C; Schnoor, Jerald L

    2017-08-12

    Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01-0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6-14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.

  16. Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement

    DEFF Research Database (Denmark)

    Kublik, Anja; Deobald, Darja; Hartwig, Stefanie

    2016-01-01

    electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250–270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster...... of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi....

  17. High frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes

    Directory of Open Access Journals (Sweden)

    Mikihiko eKawai

    2014-03-01

    Full Text Available Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5 and 107.0 mbsf at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB, key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important, functioning in the organic-rich subseafloor sedimentary biosphere.

  18. Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

    Energy Technology Data Exchange (ETDEWEB)

    Omi, Rie [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Jitsumori, Keiji; Yamauchi, Takahiro; Ichiyama, Susumu; Kurihara, Tatsuo; Esaki, Nobuyoshi [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kamiya, Nobuo; Hirotsu, Ken, E-mail: hirotsu@sci.osaka-cu.ac.jp; Miyahara, Ikuko [Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2007-07-01

    A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P6{sub 3}. Diffraction data have been collected to 1.75 Å resolution. dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6{sub 3}, with unit-cell parameters a = b = 186.2, c = 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V{sub M} value of 4.20–2.10 Å{sup 3} Da{sup −1}. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.

  19. Nitrite-mediated hydrolysis of epoxides catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 : A new tool for the kinetic resolution of epoxides

    NARCIS (Netherlands)

    Hasnaoui, Ghania; Lutje Spelberg, Jeffrey H.; de Vries, Erik; Tang, Lixia; Hauer, Bernhard; Janssen, Dick B.

    2005-01-01

    Halohydrin dehalogenase obtained from Agrobacterium radiobacter AD1, has been tested for the nitrite-mediated ring opening of epoxides. This reaction mainly leads to the formation of unstable hydroxynitrite ester intermediates, which can be further hydrolyzed to the corresponding diols. This

  20. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    Energy Technology Data Exchange (ETDEWEB)

    Schmidberger, Jason W. [School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia); Wilce, Jackie A. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); Weightman, Andrew J. [School of Biosciences, Cardiff University, Cardiff,Wales (United Kingdom); Wilce, Matthew C. J., E-mail: matthew.wilce@med.monash.edu.au [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia)

    2008-07-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2{sub 1}), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.

  1. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    International Nuclear Information System (INIS)

    Schmidberger, Jason W.; Wilce, Jackie A.; Weightman, Andrew J.; Wilce, Matthew C. J.

    2008-01-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2 1 ), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary

  2. Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

    Science.gov (United States)

    Tang, Lixia; Torres Pazmiño, Daniel E; Fraaije, Marco W; de Jong, René M; Dijkstra, Bauke W; Janssen, Dick B

    2005-05-03

    Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.

  3. Identification of Multiple Dehalogenase Genes Involved in Tetrachloroethene-to-Ethene Dechlorination in a Dehalococcoides-Dominated Enrichment Culture

    Directory of Open Access Journals (Sweden)

    Mohamed Ismaeil

    2017-01-01

    Full Text Available Chloroethenes (CEs are widespread groundwater toxicants that are reductively dechlorinated to nontoxic ethene (ETH by members of Dehalococcoides. This study established a Dehalococcoides-dominated enrichment culture (designated “YN3” that dechlorinates tetrachloroethene (PCE to ETH with high dechlorination activity, that is, complete dechlorination of 800 μM PCE to ETH within 14 days in the presence of Dehalococcoides species at 5.7±1.9×107 copies of 16S rRNA gene/mL. The metagenome of YN3 harbored 18 rdhA genes (designated YN3rdhA1–18 encoding the catalytic subunit of reductive dehalogenase (RdhA, four of which were suggested to be involved in PCE-to-ETH dechlorination based on significant increases in their transcription in response to CE addition. The predicted proteins for two of these four genes, YN3RdhA8 and YN3RdhA16, showed 94% and 97% of amino acid similarity with PceA and VcrA, which are well known to dechlorinate PCE to trichloroethene (TCE and TCE to ETH, respectively. The other two rdhAs, YN3rdhA6 and YN3rdhA12, which were never proved as rdhA for CEs, showed particularly high transcription upon addition of vinyl chloride (VC, with 75±38 and 16±8.6 mRNA copies per gene, respectively, suggesting their possible functions as novel VC-reductive dehalogenases. Moreover, metagenome data indicated the presence of three coexisting bacterial species, including novel species of the genus Bacteroides, which might promote CE dechlorination by Dehalococcoides.

  4. In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2015-01-01

    Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

  5. Structure Prediction, Molecular Dynamics Simulation and Docking Studies of D-Specific Dehalogenase from Rhizobium sp. RC1

    Directory of Open Access Journals (Sweden)

    Ismaila Yada Sudi

    2012-11-01

    Full Text Available Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD simulation and docking of D-2-chloropropionate (D-2CP, D-2-bromopropionate (D-2BP, monochloroacetate (MCA, monobromoacetate (MBA, 2,2-dichloropropionate (2,2-DCP, d,l-2,3-dichloropropionate (d,l-2,3-DCP, and 3-chloropropionate (3-CP into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25–30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.

  6. Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways

    NARCIS (Netherlands)

    Poelarends, GJ; Kulakov, LA; Larkin, MJ; van Hylckama Vlieg, Johan E.T.; Janssen, DB

    The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct

  7. Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1.

    Science.gov (United States)

    Hamid, Azzmer Azzar Abdul; Hamid, Tengku Haziyamin Tengku Abdul; Wahab, Roswanira Abdul; Huyop, Fahrul

    2015-03-01

    The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Purifying, cloning and characterizing a novel dehalogenase from Bacillus sp. GZT to enhance the biodegradation of 2,4,6-tribromophenol in water.

    Science.gov (United States)

    Liang, Zhishu; Li, Guiying; An, Taicheng

    2017-06-01

    2,4,6-Tribromophenol (TBP), an intermediate of brominated flame retardants, can easily release to environment and recalcitrant to degradation. Previously, Bacillus sp. GZT, a pure aerobic strain capable of simultaneously debrominating and mineralizing TBP, was successfully isolated by us. To further obtain a practical application and dig up its TBP degradation mechanism, a total of 46.7-fold purification of a novel dehalogenase with a final specific activity of 18.9 U mg -1 and a molecular mass of 63.4 kDa was achieved. Under optimal conditions (35 °C and 200 rpm), up to 80% degradation efficiencies were achieved within 120 min. Adding H 2 O 2 , NADPH, Mn 2+ and Mg 2+ promoted enzyme reaction effectively; while EDTA, methyl viologen, Ni 2+ , Cu 2+ , Ca 2+ and Fe 2+ strongly inhibited reaction activities. The debromination of TBP was catalyzed by the enzyme at a Km of 78 μM and a Vmax of 0.65 min -1  mg protein -1 , which indicated that this dehalogenase could specifically eliminate TBP with a high efficiency and stability. Based on MALDI-TOF/TOF analysis, the dehalogenase shared 98% identity with peptide ABC transporter substrate-binding protein. One open reading frame (ORF) encoding this peptide was found in Strain GZT genome, subjected to clone and expressed in Escherichia coli (E. coli) to characterize the encoding gene. Result showed that this recombinant strain could also remove as similar amount of TBP as Bacillus sp. GZT under the identical condition. Based on these results, we suggest that this newly-isolated TBP dehalogenase highlights a new approach for remediating TBP pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. How Many Conformations of Enzymes Should Be Sampled for DFT/MM Calculations? A Case Study of Fluoroacetate Dehalogenase

    Directory of Open Access Journals (Sweden)

    Yanwei Li

    2016-08-01

    Full Text Available The quantum mechanics/molecular mechanics (QM/MM method (e.g., density functional theory (DFT/MM is important in elucidating enzymatic mechanisms. It is indispensable to study “multiple” conformations of enzymes to get unbiased energetic and structural results. One challenging problem, however, is to determine the minimum number of conformations for DFT/MM calculations. Here, we propose two convergence criteria, namely the Boltzmann-weighted average barrier and the disproportionate effect, to tentatively address this issue. The criteria were tested by defluorination reaction catalyzed by fluoroacetate dehalogenase. The results suggest that at least 20 conformations of enzymatic residues are required for convergence using DFT/MM calculations. We also tested the correlation of energy barriers between small QM regions and big QM regions. A roughly positive correlation was found. This kind of correlation has not been reported in the literature. The correlation inspires us to propose a protocol for more efficient sampling. This saves 50% of the computational cost in our current case.

  10. Production of chlorothalonil hydrolytic dehalogenase from agro-industrial wastewater and its application in raw food cleaning.

    Science.gov (United States)

    He, Qin; Xu, Xi-Hui; Zhang, Fan; Tai, Yu-Kai; Luo, Yan-Fei; He, Jian; Hong, Qing; Jiang, Jian-Dong; Yan, Xin

    2017-06-01

    To reduce the fermentation cost for industrialization of chlorothalonil hydrolytic dehalogenase (Chd), agro-industrial wastewaters including molasses, corn steep liquor (CSL) and fermentation wastewater were used to substitute for expensive carbon and nitrogen sources and fresh water for lab preparation. The results showed that molasses and CSL could replace 5% carbon source and 100% organic nitrogen source respectively to maintain the same fermentation level. Re-fermentation from raffinate of ultra-filtered fermentation wastewater could achieve 61.03% of initial Chd activity and reach 96.39% activity when cultured in a mixture of raffinate and 50% of original medium constituent. Typical raw foods were chosen to evaluate the chlorothalonil removal ability of Chd. After Chd treatment for 2 h at room temperature, 97.40 and 75.55% of 30 mg kg -1 chlorothalonil on cherry tomato and strawberry respectively and 60.29% of 50 mg kg -1 chlorothalonil on Chinese cabbage were removed. Furthermore, the residual activity of the enzyme remained at 78-82% after treatment, suggesting its potential for reuse. This study proved the cost-feasibility of large-scale production of Chd from agro-industrial wastewater and demonstrated the potential of Chd in raw food cleaning. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. Crystallization and preliminary X-ray studies of TON-1713 from Thermococcus onnurineus NA1, a putative member of the haloacid dehalogenase superfamily

    International Nuclear Information System (INIS)

    Le, Binh Van; Lee, Hyun Sook; Cho, Yona; Kang, Sung Gyun; Kim, Dong Young; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2007-01-01

    A putative member of the haloacid dehalogenase superfamily from T. onnurineus has been expressed, purified and crystallized using 1.6 M magnesium sulfate as a precipitant. The crystals belonged to the triclinic space group P1 and diffracted to 1.8 Å resolution. The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON-1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°

  12. Identification and H(D)-bond energies of C-H(D)Cl interactions in chloride-haloalkane clusters: a combined X-ray crystallographic, spectroscopic, and theoretical study.

    Science.gov (United States)

    Serebryanskaya, Tatiyana V; Novikov, Alexander S; Gushchin, Pavel V; Haukka, Matti; Asfin, Ruslan E; Tolstoy, Peter M; Kukushkin, Vadim Yu

    2016-05-18

    The cationic (1,3,5-triazapentadiene)Pt(II) complex [Pt{NH[double bond, length as m-dash]C(N(CH2)5)N(Ph)C(NH2)[double bond, length as m-dash]NPh}2]Cl2 ([]Cl2) was crystallized from four haloalkane solvents giving [][Cl2(CDCl3)4], [][Cl2(CHBr3)4], [][Cl2(CH2Cl2)2], and [][Cl2(C2H4Cl2)2] solvates that were studied by X-ray diffraction. In the crystal structures of [][Cl2(CDCl3)4] and [][Cl2(CHBr3)4], the Cl(-) ion interacts with two haloform molecules via C-DCl(-) and C-HCl(-) contacts, thus forming the negatively charged isostructural clusters [Cl(CDCl3)2](-) and [Cl(CHBr3)2](-). In the structures of [][Cl2(CH2Cl2)2] and [][Cl2(C2H4Cl2)2], cations [](2+) are linked to a 3D-network by a system of H-bondings including one formed by each Cl(-) ion with CH2Cl2 or C2H4Cl2 molecules. The lengths and energies of these H-bonds in the chloride-haloalkane clusters were analyzed by DFT calculations (M06 functional) including AIM analysis. The crystal packing noticeably affected the geometry of the clusters, and energy of C-HCl(-) hydrogen bonds ranged from 1 to 6 kcal mol(-1). An exponential correlation (R(2) > 0.98) between the calculated Cl(-)H distances and the energies of the corresponding contacts was found and used to calculate hydrogen bond energies from the experimental Cl(-)H distances. Predicted energy values (3.3-3.9 kcal mol(-1) for the [Cl(CHCl3)2](-) cluster) are in a reasonable agreement with the energy of the Cl3C-HCl(-) bond estimated using ATRFTIR spectroscopy (2.7 kcal mol(-1)).

  13. Adaptation of Xanthobacter autotrophicus GJ10 to bromoacetate due to activation and mobilization of the haloacetate dehalogenase gene by insertion element IS1247

    DEFF Research Database (Denmark)

    van der Ploeg, J; Willemsen, M; Van Hall, Gerrit

    1995-01-01

    B gene. In mutant GJ10M50, a DNA fragment (designated IS1247) had copied itself from a position on the chromosome that was not linked to the dhlB region to a site immediately upstream of dhlB, resulting in a 1,672-bp insertion. IS1247 was found to encode an open reading frame corresponding to 464 amino...... acids which showed similarity to putative transposases from two other insertion elements. In most of the other MBA-resistant mutants of GJ10, IS1247 was also present in one more copy than in the wild type, which had two copies located within 20 kb. After insertion to a site proximal to dhlB, IS1247...... was able to transpose itself together with the dhlB gene to a plasmid, without the requirement of a second insertion element being present downstream of dhlB. The results show that IS1247 causes bromoacetate resistance by overexpression and mobilization of the haloacid dehalogenase gene, which mimics steps...

  14. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

    Directory of Open Access Journals (Sweden)

    Hui Jian

    2016-07-01

    Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

  15. Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer

    Directory of Open Access Journals (Sweden)

    Wittebolle Lieven

    2010-02-01

    Full Text Available Abstract Background Bacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ. Results We report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation. Conclusions The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time.

  16. Fluorescence-based biosensor for monitoring of environmental pollutants: From concept to field application

    Czech Academy of Sciences Publication Activity Database

    Bidmanová, Š.; Kotlanova, M.; Rataj, Tomáš; Damborský, J.; Trtílek, M.; Prokop, Z.

    2016-01-01

    Roč. 84, oct (2016), s. 97-105 ISSN 0956-5663 Grant - others:GA MŠk(CZ) LO1214 Institutional support: RVO:67179843 Keywords : dehydrochlorinase * environmental monitoring * field-testing * haloalkane dehalogenase * Halogenated pollutant * optical biosensor Subject RIV: EH - Ecology, Behaviour Impact factor: 7.780, year: 2016

  17. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis

    International Nuclear Information System (INIS)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Kuta Smatanova, Ivana

    2008-01-01

    Three mutants of the haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 were crystallized and diffracted to ultrahigh resolution. The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2 1 2 1 2 1 , while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 Å, respectively

  18. Biodegradace environmentálních polutantů - charakterizace mutantní halogenalkandehalogenasy DhaA31 z Rhodococcus rhodochrous NCIMB 13064

    OpenAIRE

    MALCHER, Pavel

    2013-01-01

    The project was focused on using of protein crystallography practically and mainly on preparation of crystals of model protein Thermolysin and haloalkan dehalogenase mutant variant DhaA31 with the use of standard and advanced crystallization methods. Later the DhaA31 structure was solved and graphically visualized using the molecular modeling programs as well as the structure of Thermolysin. Obtaining the crystals of both studied proteins and description of the DhaA31 structure, active site a...

  19. A Pseudomonas putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation

    OpenAIRE

    Samin, Ghufrana; Pavlova, Martina; Arif, M. Irfan; Postema, Christiaan P.; Damborsky, Jiri; Janssen, Dick B.

    2014-01-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP...

  20. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane

    Czech Academy of Sciences Publication Activity Database

    Lahoda, M.; Chaloupková, R.; Stsiapanava, A.; Damborský, J.; Kutá-Smatanová, Ivana

    2011-01-01

    Roč. 67, Part 3 (2011), s. 397-400 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010; GA MŠk(CZ) ED1.1.00/02.0073 Institutional research plan: CEZ:AV0Z60870520 Keywords : haloalkane dehalogenases * DhaA * Rhodococcus rhodochrous Subject RIV: CE - Biochemistry Impact factor: 0.506, year: 2011

  1. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane

    International Nuclear Information System (INIS)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-01-01

    A mutant of the haloalkane dehalogenase DhaA (DhaA31) from R. rhodochrous NCIMB 13064 and its complex with 1,2,3-trichloropropane were crystallized and the crystals diffracted to high resolution. Haloalkane dehalogenases hydrolyze carbon–halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure–function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP

  2. Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Bidmanova, Sarka; Chaloupkova, Radka; Damborsky, Jiri; Prokop, Zbynek [Masaryk University, Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Brno (Czech Republic)

    2010-11-15

    An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants. (orig.)

  3. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis.

    Science.gov (United States)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Smatanova, Ivana Kuta

    2008-02-01

    The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.

  4. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane.

    Science.gov (United States)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-03-01

    Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.

  5. Dynamics and hydration explain failed functional transformation in dehalogenase design

    Czech Academy of Sciences Publication Activity Database

    Sýkora, Jan; Brezovský, J.; Koudeláková, T.; Lahoda, M.; Fořtová, A.; Chernovets, Tatsiana; Chaloupková, R.; Štěpánková, V.; Prokop, Z.; Kutá-Smatanová, Ivana; Hof, Martin; Damborský, J.

    2014-01-01

    Roč. 10, č. 6 (2014), s. 428-430 ISSN 1552-4450 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 ; RVO:67179843 Keywords : fluorescence * dynamics * hydration Subject RIV: CF - Physical ; Theoretical Chemistry ; CE - Biochemistry (UEK-B) Impact factor: 12.996, year: 2014

  6. Purification and properties of a new dehalogenase enzyme from ...

    African Journals Online (AJOL)

    Halogenated compounds are widely used in agriculture and industries and have been associated with environmental pollution. Degradation of 3-chloropropionate (3CP) by microorganism has been established and this enzyme could only remove halogen atom at the â- position of 3-carbon alkanoic acids. Pseudomonas sp ...

  7. Dehalogenase: The Follow-Up Enzyme After Mustard Oxidation

    National Research Council Canada - National Science Library

    Elashvili, Ilya; DeFrank, Joseph J

    2002-01-01

    Sulfur mustard (HD) has been used as a chemical warfare agent since 1917. Currently fielded M258A1 and M280 decontamination kits and prospective oxidative decontaminants convert HD to HD sulfoxide (HDSO...

  8. A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

    Science.gov (United States)

    Samin, Ghufrana; Pavlova, Martina; Arif, M Irfan; Postema, Christiaan P; Damborsky, Jiri; Janssen, Dick B

    2014-09-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

    Science.gov (United States)

    Gong, Ting; Xu, Xiaoqing; Che, You; Liu, Ruihua; Gao, Weixia; Zhao, Fengjie; Yu, Huilei; Liang, Jingnan; Xu, Ping; Song, Cunjiang; Yang, Chao

    2017-08-01

    An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP + ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

  10. Balancing the Stability–Activity Trade-Off by Fine-Tuning Dehalogenase Access Tunnels

    Czech Academy of Sciences Publication Activity Database

    Lišková, V.; Bednář, D.; Prudnikova, Tatyana; Řezáčová, P.; Koudeláková, T.; Šebestová, E.; Kutá-Smatanová, Ivana; Březovský, J.; Chaloupková, R.; Damborský, J.

    2015-01-01

    Roč. 7, č. 4 (2015), s. 648-659 ISSN 1867-3880 Institutional support: RVO:67179843 Keywords : alkanes * enzymes * catalysis * halogenation * molecular dynamics * protein engineering Subject RIV: CE - Biochemistry Impact factor: 4.724, year: 2015

  11. Balancing the Stability-Activity Trade-Off by Fine-Tuning Dehalogenase Access Tunnels

    Czech Academy of Sciences Publication Activity Database

    Liskova, V.; Bednář, D.; Prudníková, T.; Řezáčová, Pavlína; Koudeláková, T.; Šebestová, E.; Smatanová, I.K.; Brezovský, J.; Chaloupková, R.

    2015-01-01

    Roč. 7, č. 4 (2015), s. 648-659 ISSN 1867-3880 Grant - others:GA ČR(CZ) GAP207/12/0775; GA MŠk(CZ) LO1214; GA MŠk(CZ) LH14027; European Social Fund(XE) CZ.1.07/2.3.00/30.0037 Program:GA Institutional support: RVO:68378050 Keywords : alkanes * halogenation * molecular dynamics * enzyme catalysis * protein engineering Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.724, year: 2015

  12. A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site

    Science.gov (United States)

    Munro, Jacob E.; Liew, Elissa F.; Ly, Mai-Anh

    2016-01-01

    ABSTRACT 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more

  13. The development and application of engineered proteins for bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [ed.

    1995-09-26

    Clean up of the toxic legacy of the Cold War is projected to be the most expensive domestic project the nation has yet undertaken. Remediation of the Department of Energy and Department of Defense toxic waste sites alone are projected to cost {approximately}$1 trillion over a 20-30 year period. New, cost effective technologies are needed to attack this enormous problem. Los Alamos has put together a cross-divisional team of scientist to develop science based bioremediation technology to work toward this goal. In the team we have expertise in: (1) molecular, ecosystem and transport modeling; (2) genetic and protein engineering; (3) microbiology and microbial ecology; (4) structural biology; and (5) bioinorganic chemistry. This document summarizes talks at a workshop of different aspects of bioremediation technology including the following: Introducing novel function into a Heme enzyme: engineering by excavation; cytochrome P-450: ideal systems for bioremediation?; selection and development of bacterial strains for in situ remediation of cholorinated solvents; genetic analysis and preparation of toluene ortho-monooxygenase for field application in remediation of trichloroethylene; microbial ecology and diversity important to bioremediation; engineering haloalkane dehalogenase for bioremediation; enzymes for oxidative biodegradation; indigenous bacteria as hosts for engineered proteins; performance of indigenous bacterial, hosting engineered proteins in microbial communities.

  14. A 13-week research-based biochemistry laboratory curriculum.

    Science.gov (United States)

    Lefurgy, Scott T; Mundorff, Emily C

    2017-09-01

    Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given. Unique to this curriculum is a successful implementation of saturation mutagenesis and high-throughput screening of enzyme function, along with bioinformatics analysis, homology modeling, structural analysis, protein expression and purification, polyacrylamide gel electrophoresis, UV/Vis spectroscopy, and enzyme kinetics. Each of these techniques is carried out using a novel student-designed mutant library or enzyme variant unique to the lab team and, importantly, not described previously in the literature. Use of a well-established set of protocols promotes student data quality. Publication may result from the original student-generated hypotheses and data, either from the class as a whole or individual students that continue their independent projects upon course completion. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):437-448, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  15. Directed evolution of enzymes using microfluidic chips

    Science.gov (United States)

    Pilát, Zdeněk.; Ježek, Jan; Šmatlo, Filip; Kaůka, Jan; Zemánek, Pavel

    2016-12-01

    Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate large variety of reactions, while ensuring appropriate catalytic activity and high selectivity. These properties make enzymes attractive biocatalysts for a wide range of industrial and biomedical applications. Over the last two decades, directed evolution of enzymes has transformed the field of protein engineering. We have devised microfluidic systems for directed evolution of haloalkane dehalogenases in emulsion droplets. In such a device, individual bacterial cells producing mutated variants of the same enzyme are encapsulated in microdroplets and supplied with a substrate. The conversion of a substrate by the enzyme produced by a single bacterium changes the pH in the droplet which is signalized by pH dependent fluorescence probe. The droplets with the highest enzymatic activity can be separated directly on the chip by dielectrophoresis and the resultant cell lineage can be used for enzyme production or for further rounds of directed evolution. This platform is applicable for fast screening of large libraries in directed evolution experiments requiring mutagenesis at multiple sites of a protein structure.

  16. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Science.gov (United States)

    Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

    2015-11-01

    There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  17. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Directory of Open Access Journals (Sweden)

    David Bednar

    2015-11-01

    Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  18. XRD and FTIR studies the effect of heat treatment and doping the transition metal oxide on LiNbO3 and LiNb3O8 nano-crystallite phases in lithium borate glass system.

    Science.gov (United States)

    Kashif, Ismail; Soliman, Ashia A; Sakr, Elham M; Ratep, Asmaa

    2013-09-01

    Glasses of various compositions in the system 90 Li2B4O7-10 Nb2O5 mixed with T.M ions (where T.M is the transition metal) were prepared by quenching technique. Heat-treatment of the parent glasses was performed at 540, 570 and 620 °C, for 5 and 16 h. The glass structure evolution during the controlled crystallization was examined by XRD and FT-IR spectroscopy analysis. The crystalline phases present in the glass ceramics were identified via X-ray diffraction as a function of heat treatment. The FT-IR data propose for these glasses and heat-treated glass network structures mainly built by: di-, tri-, tetra-, penta-and ortho-borate groups. It was found that the quantitative evolution of these various borate species in the glass structures is influenced by the transition metal. A detailed discussion relating to the N4 evolution with the T.M content was made. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.

    Science.gov (United States)

    Chovancova, Eva; Pavelka, Antonin; Benes, Petr; Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, Jiri

    2012-01-01

    Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

  20. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.

    Directory of Open Access Journals (Sweden)

    Eva Chovancova

    Full Text Available Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

  1. CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

    Science.gov (United States)

    Strnad, Ondrej; Brezovsky, Jan; Kozlikova, Barbora; Gora, Artur; Sustr, Vilem; Klvana, Martin; Medek, Petr; Biedermannova, Lada; Sochor, Jiri; Damborsky, Jiri

    2012-01-01

    Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz. PMID:23093919

  2. Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

    Science.gov (United States)

    Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

    2015-12-01

    A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

  3. Microbial dehalogenation

    NARCIS (Netherlands)

    Janssen, DB; Oppentocht, JE; Poelarends, GJ

    Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive

  4. Protein (Cyanobacteria): 652400677 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available WP_026796473.1 ... 1117:3549 ... 1150:52595 1301283:73827 ... 54304:285 54307:343 ... haloacid dehalogenase Plankt...LRDVVQKFGERLGFSPTPTELESLANSIQDWQPFPDTIAALKALKQKYKLVIISNIDDNLFAQTNQHLQIEFDHIITAQQAQSYKPSAHNFQFALNKTGLSSDKLLHVAQSIFHDIATANSLGLTTVWVNRRQGQPGGGATKAAIAQPDLEVPDLKSLVDLIFEV

  5. Novel enzymic hydrolytic dehalogenation of a chlorinated aromatic

    International Nuclear Information System (INIS)

    Scholten, J.D.; Chang, Kaihsuan; Dunaway-Mariano, D.; Babbitt, P.C.; Charest, H.; Sylvestre, M.

    1991-01-01

    Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-filobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an αβ dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide)

  6. Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

    Science.gov (United States)

    Lal, Rup; Pandey, Gunjan; Sharma, Pooja; Kumari, Kirti; Malhotra, Shweta; Pandey, Rinku; Raina, Vishakha; Kohler, Hans-Peter E.; Holliger, Christof; Jackson, Colin; Oakeshott, John G.

    2010-01-01

    Summary: Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation. PMID:20197499

  7. Photorefractive features of non-stoichiometry codoped Hf:Fe:LiNbO3 single crystals

    International Nuclear Information System (INIS)

    Liu, Bo; Li, Chunliang; Bi, Jiancong; Sun, Liang; Xu, Yuheng

    2008-01-01

    Hf(2mol%):Fe(0.05wt%):LiNbO 3 crystals with various [Li]/[Nb] ratios of 0.94, 1.05, 1.2 and 1.38 have been grown. The photorefractive resistant ability increases with the accretion of [Li]/[Nb] ratio. When the ratio of [Li]/[Nb] is 1.20 or 1.38, the OH - absorption band shifts to about 3477cm -1 . The mechanisms of the photorefractive resistant ability increase and the absorption band shift have been discussed. The exponential gain coefficient (Γ) of the crystals was measured with two-beam coupling method and the effective charge carrier concentration (N eff ) was calculated. The results show that Γ and N eff increase with the accretion of [Li]/[Nb] ratio. The temperature effect of codoped Hf:Fe:LiNbO 3 crystals was also studied, it was found that the exponential gain coefficient increase dramatically at about 55 C, 70 C and 110 C, this is due to the inner electric field which is resulted from structure phase change. (copyright 2007 WILEY -VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  8. One pot 'click' reactions : tandem enantioselective biocatalytic epoxide ring opening and [3+2] azide alkyne cycloaddition

    NARCIS (Netherlands)

    Campbell-Verduyn, Lachlan S.; Szymanski, Wiktor; Postema, Christiaan P.; Dierckx, Rudi A.; Elsinga, Philip H.; Janssen, Dick B.; Feringa, Ben L.

    2010-01-01

    Halohydrin dehalogenase (HheC) can perform enantioselective azidolysis of aromatic epoxides to 1,2-azido alcohols which are subsequently ligated to alkynes producing chiral hydroxy triazoles in a one-pot procedure with excellent enantiomeric excess.

  9. Primers That Target Functional Genes of Organohalide-Respiring Bacteria (online first)

    NARCIS (Netherlands)

    Lu, Y.; Atashgahi, S.; Hug, L.A.; Smidt, H.

    2015-01-01

    Halogenated organic hydrocarbons are problematic environmental pollutants that can be reductively dehalogenated by organohalide-respiring bacteria (OHRB) in anoxic environments. This energy-conserving process is mediated by reductive dehalogenases (RDases). To amplify the diversity of reductive

  10. Evaluating iodide recycling inhibition as a novel molecular initiating event for thyroid axis disruption

    Science.gov (United States)

    The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces available iodide for thyroid hormone synthesis, which leads to hormone insufficiency...

  11. A tiered approach to evaluate an iodine recycling inhibition adverse outcome pathway (AOP) in amphibians

    Science.gov (United States)

    The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces thyroid hormone synthesis, which leads to insufficiency in tissues and subsequent ne...

  12. Microscope Raman scattering and X-ray diffraction study of near-stoichiometric Ti:LiNbO3 waveguides

    International Nuclear Information System (INIS)

    Zhang, De-Long; Siu, G.G.; Pun, E.Y.B.

    2005-01-01

    The crystalline phase within guiding layers of near-stoichiometric strip and planar Ti:LiNbO 3 wave-guides, prepared by the method of simultaneous work of vapour transport equilibration (VTE) treatment and indiffusion of Ti film, was studied by combined confocal microscope Raman scattering and X-ray powder diffraction. The results show that the strip and planar waveguide layers still retain the LiNbO 3 phase and no other non-LiNbO 3 phases can be identified within the guiding layer. Li/Nb ratios inside and outside the strip and planar waveguide layers were determined from the microscope Raman scattering results and compared to those obtained from the measured optical absorption edge. It is shown that the Li/Nb ratios are homogeneous within the waveguide layer and are close inside and outside the waveguide layer. (copyright 2005 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  13. Unstable Resonator Retrofitted Handheld Laser Designator

    Science.gov (United States)

    1978-06-01

    retrofitted with a negative-branch unstable resona- tor laser and hybrid pump cavity in place of the conventional plane-mirror/ porro prism resonator and...directed by prism B to an expanding telescope, shared with the viewing system of the designator. The actual, unfolded resonator length is approxi...was performed based on using a plane- parallel cavity consisting of a 47% reflectivity output coupler, porro - prism reflector, and the same LiNb03

  14. Biochemistry of Catabolic Reductive Dehalogenation.

    Science.gov (United States)

    Fincker, Maeva; Spormann, Alfred M

    2017-06-20

    A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

  15. Partial hydration of n-alkyl halides at the water-vapor interface: a molecular simulation study with atmospheric implications

    Czech Academy of Sciences Publication Activity Database

    Habartová, Alena; Obisesan, A.; Minofar, Babak; Roeselová, Martina

    2014-01-01

    Roč. 133, č. 3 (2014), 1455/1-1455/15 ISSN 1432-881X R&D Projects: GA ČR(CZ) GAP208/10/1724; GA MŠk ME09064 Grant - others:GA ČR(CZ) GA13-08651S Institutional support: RVO:61388963 ; RVO:67179843 Keywords : haloalkanes * adsorption * interfacial solvation * halogenated organics * interfacial partitioning * surface orientation * heterogeneous chemistry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.233, year: 2014

  16. Conversion of broadband IR radiation and structural disorder in lithium niobate single crystals with low photorefractive effect

    Science.gov (United States)

    Litvinova, Man Nen; Syuy, Alexander V.; Krishtop, Victor V.; Pogodina, Veronika A.; Ponomarchuk, Yulia V.; Sidorov, Nikolay V.; Gabain, Aleksei A.; Palatnikov, Mikhail N.; Litvinov, Vladimir A.

    2016-11-01

    The conversion of broadband IR radiation when the noncritical phase matching condition is fulfilled in lithium niobate (LiNbO3) single crystals with stoichiometric (R = Li/Nb = 1) and congruent (R = 0.946) compositions, as well as in congruent single crystals doped with zinc has been investigated. It is shown that the spectrum parameters of converted radiation, such as the conversion efficiency, spectral width and position of maximum, depend on the ordering degree of structural units of the cation sublattice along the polar axis of crystal.

  17. Investigation of the cluster formation in lithium niobate crystals by computer modeling method

    Energy Technology Data Exchange (ETDEWEB)

    Voskresenskii, V. M.; Starodub, O. R., E-mail: ol-star@mail.ru; Sidorov, N. V.; Palatnikov, M. N. [Russian Academy of Sciences, Tananaev Institute of Chemistry and Technology of Rare Earth Elements and Mineral Raw Materials, Kola Science Centre (Russian Federation)

    2017-03-15

    The processes occurring upon the formation of energetically equilibrium oxygen-octahedral clusters in the ferroelectric phase of a stoichiometric lithium niobate (LiNbO{sub 3}) crystal have been investigated by the computer modeling method within the semiclassical atomistic model. An energetically favorable cluster size (at which a structure similar to that of a congruent crystal is organized) is shown to exist. A stoichiometric cluster cannot exist because of the electroneutrality loss. The most energetically favorable cluster is that with a Li/Nb ratio of about 0.945, a value close to the lithium-to-niobium ratio for a congruent crystal.

  18. Experimental and theoretical investigation of lattice defect structures in a series of Zn, Fe-doped nonstoichiometric lithium niobate

    International Nuclear Information System (INIS)

    Guo Fengyun; Lue Qiang; Sun Liang; Li Hongtao; Zhen Xihe; Xu Yuheng; Zhao Liancheng

    2006-01-01

    A series of the double doped lithium niobate (LiNbO 3 , LN) single crystals had been grown by Czochralski method. The Curie temperatures of various concentrations doped or [Li]/[Nb] ratio LN crystals measured by differential thermal analysis (DTA) were discussed to investigate their defect structures with Safaryan et al. new approach about LN lattice defect structure using Curie temperatures calculated. Infrared transmission spectra of various concentrations doped were used to compare the investigation above. The results show that the lithium vacancy model is the more probable to describe the lattice defect structure of the doped LN single crystal

  19. Preparação de LiNbO3 e LiNbO3:Eu3+ pelo método dos precursores poliméricos

    Directory of Open Access Journals (Sweden)

    Souza Luciana A.

    2002-01-01

    Full Text Available The ferroelectric compound LiNbO3 was prepared by the polymeric precursors method in the polycrystalline form containing different concentrations of Eu3+. The compounds were characterized by X-ray diffraction, vibrational and electronic spectroscopy. Electronic spectroscopy was shown to be sensitive to small concentrations of contaminating phases allowing a good control of the compound purity. The presence of Eu3+ ions leads to the formation of the LiNb3O8 phase in the range of 500 to 800 degreesC. Above this temperature range LiNbO3 and tetragonal (T' EuNbO4 were obtained.

  20. Cloning of 1,2-Dichloroethane Degradation Genes of Xanthobacter autotrophicus GJ10 and Expression and Sequencing of the dhlA Gene

    NARCIS (Netherlands)

    Janssen, Dick B.; Pries, Frens; Ploeg, Jan van der; Kazemier, Bert; Terpstra, Peter; Witholt, Bernard

    1989-01-01

    A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in

  1. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences. Dhurairajan senthilnathan. Articles written in Journal of Chemical Sciences. Volume 123 Issue 3 May 2011 pp 279-290. Biocatalysis of azidolysis of epoxides: Computational evidences on the role of halohydrin dehalogenase (HheC) · Dhurairajan senthilnathan ...

  2. Nanostructured sodium lithium niobate and lithium niobium tantalate solid solutions obtained by controlled crystallization of glass

    International Nuclear Information System (INIS)

    Radonjic, L.; Todorovic, M.; Miladinovic, J.

    2005-01-01

    Transparent, nanostructured glass ceramics based on ferroelectric solid solutions of the type Na 1-x Li x NbO 3 (in very narrow composition regions for x = 0.12 and 0.93) and LiNb 1-y Ta y O 3 (y = 0.5 unlimited solid solubility), can be obtained by controlled crystallization of glass. The parent glass samples were prepared by conventional melt-quenching technique. Heat-treatment of the parent glasses was performed at the various temperatures, for the same time. The glass structure evolution during the controlled crystallization was examined by FT-IR spectroscopy analysis. Crystalline phases were identified by X-ray diffraction analysis and SEM was used for microstructure characterization. Densities of the crystallized glasses were measured by Archimedean principle. The capacitance and dielectric loss tangent were measured at a frequency of 1 kHz, at the room temperature. It was found that in the all investigated systems crystallize solid solutions Na 1-x Li x NbO 3 and LiNb 1-y Ta y O 3 in the glassy matrix, have crystal size on nanoscale (less than 100 nm), which is one of requirements to get a transparent glass ceramic that could be a good ferroelectric material regarding to the measured properties

  3. The effect of Mg doping on the Raman spectra of LiNbO3 crystals

    International Nuclear Information System (INIS)

    Lengyel, K.; Kovacs, L.; Peter, A.; Polgar, K.; Corradi, G.; Bourson, P.

    2007-01-01

    LiNbO 3 :Mg crystals doped with 0-8 mol% Mg with stoichiometric, intermediate and congruent compositions were systematically investigated by Raman spectroscopy in backscattering y(zx)y and y(zz)y geometries. The halfwidths of E(TO 3 ) - E(TO 9 ) and A 1 (TO 1 ) - A 1 (TO 4 ) bands, which have a significant composition dependence for undoped LiNbO 3 crystals, show only weak Mg-concentration dependence below the photorefractive threshold. This is a consequence of the counteracting effect of the decreasing amount of Nb ions at Li-ion sites (Nb Li ) and increasing amount of Mg ions at Li-ion sites (Mg Li ). The halfwidths of these bands, however, increase linearly with growing Mg content for samples above the threshold, irrespective of the Li/Nb ratio. The same effect was observed in the Mg dependence of the position and halfwidth of the two-phonon band at about 689 - 705 cm -1 . The change in the Mg concentration dependence of these parameters at a given Li/Nb ratio determines the same threshold value as that concluded from IR and UV spectroscopic measurements. (copyright 2007 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  4. Synthesis and Side Chain Liquid Crystal Polymers by Living Ring Opening Metathesis Polymerization. 5. Influence of Mesogenic Group and Interconnecting Group on the Thermotropic Behavior of the Resulting Polymers

    Science.gov (United States)

    1992-07-22

    Scheme I. The first nucleophilic displacement of halide of an n-haloalkan-l-ol with 4-cyano-4’-hydroxybiphenyl employed potassium carbonate in...21 𔃼 polysiloxanes, 23.24 and polyacrylates . 2- All these polymers exhibit an odd-even effect. If one considers the total number of atoms between the...0.019 mol) and 4’-methoxy-4-hydroxybiphenyl (4.0g, 0.020 tool) were heated at 100°C in 40 mL of dimethylformamide in the presence of potassium carbonate

  5. Biominerlization and possible endosulfan degradation pathway adapted by Aspergillus niger.

    Science.gov (United States)

    Bhalerao, Tejomyee S

    2013-11-28

    Endosulfan is a chlorinated pesticide; its persistence in the environment and toxic effects on biota are demanding its removal. This study aims at improving the tolerance of the previously isolated fungus Aspergillus niger (A. niger) ARIFCC 1053 to endosulfan. Released chloride, dehalogenase activity, and released proteins were estimated along with analysis of endosulfan degradation and pathway identification. The culture could tolerate 1,000 mg/ml of technical grade endosulfan. Complete disappearance of endosulfan was seen after 168 h of incubation. The degradation study could easily be correlated with increase in released chlorides, dehalogenase activity and protein released. Comparative infrared spectral analysis suggested that the molecule of endosulfan was degraded efficiently by A. niger ARIFCC 1053. Obtained mass ion values by GC-MS suggested a hypothetical pathway during endosulfan degradation by A. niger ARIFCC 1053. All these results provide a basis for the development of bioremediation strategies to remediate the pollutant under study in the environment.

  6. Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans

    International Nuclear Information System (INIS)

    Feng, Ming-Jing; Fu, Tian-Min; Liu, Xiang; Li, Lan-Fen

    2010-01-01

    SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å. Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°

  7. Cation-Specific Effects on Enzymatic Catalysis Driven by Interactions at the Tunnel Mouth

    Czech Academy of Sciences Publication Activity Database

    Štěpánková, V.; Paterová, Jana; Damborský, J.; Jungwirth, Pavel; Chaloupková, R.; Heyda, Jan

    2013-01-01

    Roč. 117, č. 21 (2013), s. 6394-6402 ISSN 1520-6106 R&D Projects: GA ČR GBP208/12/G016 Grant - others:GA ČR(CZ) GAP207/12/0775; GA MŠk(CZ) ED1.100/02/0123 Program:GA Institutional support: RVO:61388963 Keywords : dehalogenase * Hofmeister series * enzyme kinetics * molecular dynamics Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

  8. Enhanced degradation of haloacid by heterologous expression in related Burkholderia species.

    Science.gov (United States)

    Su, Xianbin; Deng, Liyu; Kong, Ka Fai; Tsang, Jimmy S H

    2013-10-01

    Haloacids are environmental pollutant and can be transformed to non-toxic alkanoic acids by microbial dehalogenase. Bacterium Burkholderia species MBA4 was enriched from soil for its ability to bioremediate haloacids such as mono-chloroacetate (MCA), mono-bromoacetate (MBA), 2-mono-chloropropionate, and 2-mono-bromopropionate. MBA4 produces an inducible dehalogenase Deh4a that catalyzes the dehalogenation process. The growth of MBA4 on haloacid also relies on the presence of a haloacid-uptake system. Similar dehalogenase genes can be found in the genome of many related species. However, wildtype Burkholderia caribensis MWAP64, Burkholderia phymatum STM815, and Burkholderia xenovorans LB400 were not able to grow on MCA. When a plasmid containing the regulatory and structural gene of Deh4a was transformed to these species, they were able to grow on haloacid. The specific enzyme activities in these recombinants ranges from 2- to 30-fold that of MBA4 in similar condition. Reverse transcription-quantitative real-time PCR showed that the relative transcript levels in these recombinant strains ranges from 9 to over 1,600 times that of MBA4 in similar condition. A recombinant has produced nearly five times of dehalogenase that MBA4 could ever achieve. While the expressions of Deh4a were more relaxed in these phylogenetically related species, an MCA-uptake activity was found to be inducible. These metabolically engineered strains are better degraders than the haloacid-enriched MBA4. Copyright © 2013 Wiley Periodicals, Inc.

  9. Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

    Science.gov (United States)

    Mukherjee, Kalpataru; Bowman, Kimberly S; Rainey, Fred A; Siddaramappa, Shivakumara; Challacombe, Jean F; Moe, William M

    2014-05-01

    The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. LiNbO3 :Pr3+ : A Multipiezo Material with Simultaneous Piezoelectricity and Sensitive Piezoluminescence.

    Science.gov (United States)

    Tu, Dong; Xu, Chao-Nan; Yoshida, Akihito; Fujihala, Masayoshi; Hirotsu, Jou; Zheng, Xu-Guang

    2017-06-01

    Red-emitting piezoluminescence (elasticoluminescence) is achieved by doping rare earth Pr 3+ into the well-known piezoelectric matrix, LiNbO 3 . By precisely tuning the Li/Nb ratio in nonstoichiometric Li x NbO 3 :Pr 3+ , a material that exhibits an unusually high piezoluminescence intensity, which far exceeds that of any well-known piezoelectric material, is produced. Li x NbO 3 :Pr 3+ shows excellent strain sensitivity at the lowest strain level, with no threshold for stress sensing. These multipiezo properties of sensitive piezoluminescence in a piezoelectric matrix are ideal for microstress sensing, damage diagnosis, electro-mechano-optical energy conversion, and multifunctional control in optoelectronics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Conversion of broadband thermal radiation in lithium niobate crystals of various compositions

    Science.gov (United States)

    Syuy, A. V.; Litvinova, M. N.; Goncharova, P. S.; Sidorov, N. V.; Palatnikov, M. N.; Krishtop, V. V.; Likhtin, V. V.

    2013-05-01

    The conversion of the broadband thermal radiation in stoichiometric ( R = 1) lithium niobate single crystals that are grown from melt with 58.6 mol % of LiO2, congruent ( R = Li/Nb = 0.946) melt with the K2O flux admixture (4.5 and 6.0 wt %), and congruent melt and in congruent single crystals doped with the Zn2+, Gd3+, and Er3+ cations is studied. It is demonstrated that the conversion efficiency of the stoichiometric crystal that is grown from the melt with 58.6 mol % of LiO2 is less than the conversion efficiency of congruent crystal. In addition, the stoichiometric and almost stoichiometric crystals and the doped congruent crystals exhibit the blue shift of the peak conversion intensity in comparison with a nominally pure congruent crystal. For the congruent crystals, the conversion intensities peak at 520 and 495 nm, respectively.

  12. Growth and holographic data storage properties of near-stoichiometric LiTaO{sub 3} crystals doped with Mn

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Tao [College of Science, Harbin Engineering University, Harbin 150001 (China)], E-mail: tzhang_hit02@yahoo.com; Dong Yantang; Geng Tao; Dai Qiang [College of Science, Harbin Engineering University, Harbin 150001 (China); Xu Yuheng [School of Astronautics, Harbin Institute of Technology, Harbin 150001 (China)

    2009-03-15

    A series of Mn-doped near-stoichiometric LiTaO{sub 3} crystals were grown from a Li-rich (Li/Nb = 1.38, atomic ratio) and varying level of Mn-doping melt using a Cz furnace equipped with a radio frequency generator. The etching experiment reveals that as-grown polarized Mn:SLN has single ferroelectric domain structures under optical microscope. By two-beam coupling experiment, we measured and systematically analyzed the photorefractive properties such as the dynamic range, the sensitivity and the loss of signal-to-noise-ratio coefficient. Based on Mn (0.05 wt%):SLN crystal, a big capacity storage of 100 holograms in a coherent volume of 0.085 cm{sup 3} have been fulfilled successfully and the storage density arrived 0.93 Gbits cm{sup -3}.

  13. Role of sintering time, crystalline phases and symmetry in the piezoelectric properties of lead-free KNN-modified ceramics

    International Nuclear Information System (INIS)

    Rubio-Marcos, F.; Marchet, P.; Merle-Mejean, T.; Fernandez, J.F.

    2010-01-01

    Lead-free KNN-modified piezoceramics of the system (Li,Na,K)(Nb,Ta,Sb)O 3 were prepared by conventional solid-state sintering. The X-ray diffraction patterns revealed a perovskite phase, together with some minor secondary phase, which was assigned to K 3 LiNb 6 O 17 , tetragonal tungsten-bronze (TTB). A structural evolution toward a pure tetragonal structure with the increasing sintering time was observed, associated with the decrease of TTB phase. A correlation between higher tetragonality and higher piezoelectric response was clearly evidenced. Contrary to the case of the LiTaO 3 modified KNN, very large abnormal grains with TTB structure were not detected. As a consequence, the simultaneous modification by tantalum and antimony seems to induce during sintering a different behaviour from the one of LiTaO 3 modified KNN.

  14. Concurrent Haloalkanoate Degradation and Chlorate Reduction by Pseudomonas chloritidismutans AW-1T.

    Science.gov (United States)

    Peng, Peng; Zheng, Ying; Koehorst, Jasper J; Schaap, Peter J; Stams, Alfons J M; Smidt, Hauke; Atashgahi, Siavash

    2017-06-15

    Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1 T , a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI , encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1 T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1 T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1 T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1 T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1 T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle

  15. The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular

    Directory of Open Access Journals (Sweden)

    Katja Seidel

    2018-06-01

    Full Text Available Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80 and its membrane anchor protein RdhB (CbdbB3. We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory

  16. [Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane].

    Science.gov (United States)

    Ziakun, A M; Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

    2007-01-01

    Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.

  17. Radiation-induced tritium labelling and product analysis

    Energy Technology Data Exchange (ETDEWEB)

    Peng, C.T. (California Univ., San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry)

    1993-05-01

    By-products formed in radiation-induced tritium labelling are identified by co-chromatography with authentic samples or by structure prediction using a quantitative structure-retention index relationship. The by-products, formed from labelling of steroids, polynuclear aromatic hydrocarbons, 7-membered heterocyclic ring structures, 1,4-benzodiazepines, 1-haloalkanes, etc. with activated tritium and adsorbed tritium, are shown to be specifically labelled and anticipated products from known chemical reactions. From analyses of the by-products, one can conclude that the hydrogen abstraction by tritium atoms and the substitution by tritium ions are the mechanisms of labelling. Classification of the tritium labelling methods, on the basis of the type of tritium reagent, clearly shows the active role played by tritium atoms and ions in radiation-induced methods. (author).

  18. Thermophilic archaeal enzymes and applications in biocatalysis.

    Science.gov (United States)

    Littlechild, Jennifer A

    2011-01-01

    Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

  19. Isolation and Identification of Sodium Fluoroacetate Degrading Bacteria from Caprine Rumen in Brazil

    Directory of Open Access Journals (Sweden)

    Expedito K. A. Camboim

    2012-01-01

    Full Text Available The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100 μL in mineral medium added with 20 mmol L−1 sodium fluoroacetate (SF, incubated at 39°C in an orbital shaker. Pseudomonas fluorescens (strain DSM 8341 was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08 and Ancylobacter dichloromethanicus (ECPB09. These bacteria degraded sodium fluoroacetate, releasing 20 mmol L−1 of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L−1 of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.

  20. Anaerobic reductive dechlorination of tetrachloroethene: how can dual Carbon-Chlorine isotopic measurements help elucidating the underlying reaction mechanism?

    Science.gov (United States)

    Badin, Alice; Buttet, Géraldine; Maillard, Julien; Holliger, Christof; Hunkeler, Daniel

    2014-05-01

    Chlorinated ethenes (CEs) such as tetrachloroethene (PCE) are common persistent groundwater contaminants. Among clean-up strategies applied to sites affected by such pollution, bioremediation has been considered with a growing interest as it represents a cost-effective, environmental friendly approach. This technique however sometimes leads to an incomplete and slow biodegradation of CEs resulting in an accumulation of toxic metabolites. Understanding the reaction mechanisms underlying anaerobic reductive dechlorination would thus help assessing PCE biodegradation in polluted sites. Stable isotope analysis can provide insight into reaction mechanisms. For chlorinated hydrocarbons, carbon (C) and chlorine (Cl) isotope data (δ13C and δ37Cl) tend to show a linear correlation with a slope (m ≡ ɛC/ɛCl) characteristic of the reaction mechanism [1]. This study hence aims at exploring the potential of a dual C-Cl isotope approach in the determination of the reaction mechanisms involved in PCE reductive dechlorination. C and Cl isotope fractionation were investigated during anaerobic PCE dechlorination by two bacterial consortia containing members of the Sulfurospirillum genus. The specificity in these consortia resides in the fact that they each conduct PCE reductive dechlorination catalysed by one different reductive dehalogenase, i.e. PceADCE which yields trichloroethene (TCE) and cis-dichloroethene (cDCE), and PceATCE which yields TCE only. The bulk C isotope enrichment factors were -3.6±0.3 o for PceATCE and -0.7±0.1o for PceADCE. The bulk Cl isotope enrichment factors were -1.3±0.2 o for PceATCE and -0.9±0.1 o for PceADCE. When applying the dual isotope approach, two m values of 2.7±0.1 and 0.7±0.2 were obtained for the reductive dehalogenases PceATCE and PceADCE, respectively. These results suggest that PCE can be degraded according to two different mechanisms. Furthermore, despite their highly similar protein sequences, each reductive dehalogenase seems

  1. Thermal plasma fabricated lithium niobate-tantalate films on sapphire substrate

    International Nuclear Information System (INIS)

    Kulinich, S.A.; Yoshida, T.; Yamamoto, H.; Terashima, K.

    2003-01-01

    We report the deposition of LiNb 1-x Ta x O 3 (0≤x≤1) films on (001) sapphire substrates in soft vacuum using a radio frequency thermal plasma. The growth rate, crystallinity, c-axis orientation, and surface roughness were examined as functions of substrate temperature, precursor feed rate, and substrate surface condition. The film Nb/Ta ratio was well controlled by using an appropriate uniform mixture of lithium-niobium and lithium-tantalum alkoxide solutions. The epitaxy and crystallinity of the films were much improved when the film growth rate was raised from 20 to 180-380 nm/min, where the films with the (006) rocking curve full width at half maximum values as low as 0.12 deg. -0.2 deg. could be produced. The film roughness could be reduced by using a liquid precursor with higher metal concentrations, achieving the root-mean-square value on the order of 5 nm. The refractive indices of the films are in good correspondence with their composition and crystallinity

  2. Molecular Beam Epitaxy of lithium niobium oxide multifunctional materials

    Science.gov (United States)

    Tellekamp, M. Brooks; Shank, Joshua C.; Doolittle, W. Alan

    2017-04-01

    The role of stoichiometry and growth temperature in the preferential nucleation of material phases in the Li-Nb-O family are explored yielding an empirical growth phase diagram. It is shown that while single parameter variation often produces multi-phase films, combining substrate temperature control with the previously published lithium flux limited growth allows the repeatable growth of high quality single crystalline films of many different oxide phases. Higher temperatures (800-1050 °C) than normally used in MBE were necessary to achieve high quality materials. At these temperatures the desorption of surface species is shown to play an important role in film composition. Using this method single phase films of NbO, NbO2, LiNbO2, Li3NbO4, LiNbO3, and LiNb3O8 have been achieved in the same growth system, all on c-plane sapphire. Finally, the future of these films in functional oxide heterostructures is briefly discussed.

  3. Influence of heat treatment on structure and some physical properties of lithium boro-niobate glass

    Science.gov (United States)

    Kashif, I.; Sakr, E. M.; Soliman, A. A.; Ratep, A.

    2012-08-01

    The glass composition (90 mol% Li2B4O7-10 mol% Nb2O5) was prepared by the melt quenching technique. The quenched sample was heat treated at 480°C, 545°C and 630°C for 5 h and heat treated at 780°C with different time. The times were 5, 10, 15, 20, 28, and 36 h. The glass and glass ceramics were studied by differential thermal analysis (DTA), X-ray diffraction (XRD), and dc conductivity as a function of temperature. Lithium niobate (LiNbO3) and lithium diborate (Li2B4O7) were the main phases in glass ceramic addition to traces from LiNb3O8. Crystallite size of the main phases determined from the X-ray diffraction peaks are in the range <100 nm. The fraction of crystalline (LiNbO3) phase increases with increase the heat treatment temperature and time. The relation between physical properties and structure were studied.

  4. Improvements of uniformity and stoichiometry for zone-leveling Czochralski growth of MgO-doped LiNbO3 crystals

    International Nuclear Information System (INIS)

    Tsai, C.B.; Hsu, W.T.; Shih, M.D.; Tai, C.Y.; Hsieh, C.K.; Hsu, W.C.; Hsu, R.T.; Lan, C.W.

    2006-01-01

    The zone-leveling Czochralski (ZLCz) technique is a continuous feeding process and can be used for the growth of near-stoichiometric lithium niobate (SLN) single crystals. However, the finite crucible length can cause the variation of the zone length and thus the composition and stoichiometry, especially in the growth of a large diameter crystal. To solve the problems, several approaches were proposed for the growth of 4 cm-diameter 1 mol% MgO-doped SLN. The modification of the hot zone to minimize the zone variation was found useful for the uniformity, but the stoichiometry was inadequate even with the zone composition up to 60 mol% Li 2 O. A Li-excess feed was further used and a good Li/Nb ratio was obtained. Adding K 2 O (16 mol%) into the solution zone was useful as well, but it was inferior to using the Li-excess feed. In addition, a much lower growth rate was needed for getting an inclusion-free crystal

  5. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    Directory of Open Access Journals (Sweden)

    Shuiquan eTang

    2016-02-01

    Full Text Available The genomes of two closely related Dehalobacter strains (strain CF and strain DCA were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF, 1,1,1-trichloroethane (1,1,1-TCA and 1,1-dichloroethane (1,1-DCA. The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA and strain DCA (that dechlorinates 1,1-DCA each contain 17 putative reductive dehalogenase homologous (rdh genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB, and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and greater than 99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1 and corrinoid biosynthesis pathways (strains E1 and PER-K23. The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to Dehalococcoides mccartyi

  6. Genome sequence of the organohalide-respiring Dehalogenimonas alkenigignens type strain (IP3-3(T)).

    Science.gov (United States)

    Key, Trent A; Richmond, Dray P; Bowman, Kimberly S; Cho, Yong-Joon; Chun, Jongsik; da Costa, Milton S; Rainey, Fred A; Moe, William M

    2016-01-01

    Dehalogenimonas alkenigignens IP3-3(T) is a strictly anaerobic, mesophilic, Gram negative staining bacterium that grows by organohalide respiration, coupling the oxidation of H2 to the reductive dehalogenation of polychlorinated alkanes. Growth has not been observed with any non-polyhalogenated alkane electron acceptors. Here we describe the features of strain IP3-3(T) together with genome sequence information and its annotation. The 1,849,792 bp high-quality-draft genome contains 1936 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small unit rRNA (16S) locus. The genome contains 29 predicted reductive dehalogenase genes, a large majority of which lack cognate genes encoding membrane anchoring proteins.

  7. [Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

    Science.gov (United States)

    Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

    2005-01-01

    Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

  8. [Adaptation of aerobic methylobacteria to dichloromethane degradation].

    Science.gov (United States)

    Torgonskaia, M L; Firsova, Iu E; Doronina, N V; Trotsenko, Iu A

    2007-01-01

    A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.

  9. Phytoremediation of small organic contaminants using transgenic plants

    Science.gov (United States)

    James, C Andrew; Strand, Stuart E

    2010-01-01

    The efficacy of transgenic plants in the phytoremediation of small organic contaminants has been investigated. Two principal strategies have been pursued (1) the manipulation of phase I metabolic activity to enhance in planta degradation rates, or to impart novel metabolic activity, and (2) the enhanced secretion of reactive enzymes from roots leading to accelerated ex planta degradation of organic contaminants. A pair of dehalogenase genes from Xanthobacter autotrophicus was expressed in tobacco resulting in the dehalogenation of 1,2-dichloroethane, which was otherwise recalcitrant. A laccase gene from cotton was overexpressed in Arabidopsis thaliana resulting in increased secretory laccase activity and the enhanced resistance to trichlorophenol in soils. Although the results to date are promising, much of the work has been limited to laboratory settings; field demonstrations are needed. PMID:19342219

  10. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  11. Anaerobic microbial dehalogenation of organohalides-state of the art and remediation strategies.

    Science.gov (United States)

    Nijenhuis, Ivonne; Kuntze, Kevin

    2016-04-01

    Contamination and remediation of groundwater with halogenated organics and understanding of involved microbial reactions still poses a challenge. Over the last years, research in anaerobic microbial dehalogenation has advanced in many aspects providing information about the reaction, physiology of microorganisms as well as approaches to investigate the activity of microorganisms in situ. Recently published crystal structures of reductive dehalogenases (Rdh), heterologous expression systems and advanced analytical, proteomic and stable isotope approaches allow addressing the overall reaction and specific enzymes as well as co-factors involved during anaerobic microbial dehalogenation. In addition to Dehalococcoides spp., Dehalobacter and Dehalogenimonas strains have been recognized as important and versatile organohalide respirers. Together, these provide perspectives for integrated concepts allowing to improve and monitor in situ biodegradation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Topological variation in the evolution of new reactions in functionally diverse enzyme superfamilies.

    Science.gov (United States)

    Meng, Elaine C; Babbitt, Patricia C

    2011-06-01

    In functionally diverse enzyme superfamilies (SFs), conserved structural and active site features reflect catalytic capabilities 'hard-wired' in each SF architecture. Overlaid on this foundation, evolutionary changes in active site machinery, structural topology and other aspects of structural organization and interactions support the emergence of new reactions, mechanisms, and substrate specificity. This review connects topological with functional variation in each of the haloalkanoic acid dehalogenase (HAD) and vicinal oxygen chelate fold (VOC) SFs and a set of redox-active thioredoxin (Trx)-fold SFs to illustrate a few of the varied themes nature has used to evolve new functions from a limited set of structural scaffolds. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. An enzymatic method for determination of azide and cyanide in aqueous phase.

    Science.gov (United States)

    Wan, Nan-Wei; Liu, Zhi-Qiang; Xue, Feng; Zheng, Yu-Guo

    2015-11-20

    A halohydrin dehalogenase (HHDH-PL) from Parvibaculum lavamentivorans DS-1 was characterized and applied to determine azide and cyanide in the water. In this methodology, HHDH-PL catalysed azide and cyanide to react with butylene oxide and form corresponding β-substituted alcohols 1-azidobutan-2-ol (ABO) and 3-hydroxypentanenitrile (HPN) that could be quantitatively detected by gas chromatograph. The detection calibration curves for azide (R(2)=0.997) and cyanide (R(2)=0.995) were linear and the lower limits of detection for azide and cyanide were 0.1 and 0.3mM, respectively. Several other nucleophiles were identified having no effect on the analysis of azide and cyanide, excepting nitrite which influenced the detection of cyanide. This was the first report of a biological method to determine the inorganic azide and cyanide by converting them to the measurable organics. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Anaerobic degradation of tetrachloroethylene; Anaerober Abbau von Tetrachlorethylen

    Energy Technology Data Exchange (ETDEWEB)

    Diekert, G [Stuttgart Univ. (Germany). Inst. fuer Mikrobiologie; Scholz-Muramatsu, H [Stuttgart Univ. (Germany). Inst. fuer Siedlungswasserbau

    1997-12-31

    Dehalospirillum multivorans, a tetrachloroethylene-dechlorinating bacterium, was isolated in activated sludge. This organism is able to grow on a defined medium with hydrogen and tetrachloroethylene (PCE) as its only energy source. The organism was characterised and the physiology of dechlorination was studied. In this process PCE is dechlorinated to cis-1,2-dichloroethene (DCE) via trichloroethene (TCE). A fluidized-bed reactor which reduces PCE to DCE at a high rate (15 nmol/min/mg of protein at 5 {mu}M PCE) was inoculated with the bacterium. Meanwhile a reactor inoculated with D. multivorans and a fully dechlorinating mixed culture has become available which catalyses the complete dechlorination of PCE to ethene at just as high rates. Tetrachloroethene dehalogenase was purified from D. multivorans (unpublished results) and characterised. (orig./SR) [Deutsch] Aus Belebtschlamm wurde ein Tetrachlorethen-dechlorierendes Bakterium, Dehalospirillum multivorans, isoliert. Der Organismus waechst auf definiertem Medium mit Wasserstoff und Tetrachlorethen (PCE) als einziger Energiequelle. Der Organismus wurde charakterisiert und die Physiologie der Dechlorierung wurde untersucht. PCE wird dabei ueber Trichlorethen (TCE) bis zum cis-1,2-Dichlorethen (DCE) dechloriert. Mit diesem Bakterium wurde ein Wirbelschichtreaktor inokuliert, der mit hohen Raten (15 nmol/min/mg Protein bei 5 {mu}M PCE) PCE zu DCE reduziert. Inzwischen steht ein Reaktor zur Verfuegung, der mit D. multivorans und einer voellig dechlorierenden Mischkultur inokuliert wurde und der mit ebenso hohen Raten eine vollstaendige Dechlorierung von PCE bis zum Ethen katalysiert. Aus D. multivorans wurde die Tetrachlorethen-Dehalogenase gereinigt (unveroeffentlichte Ergebnisse) und charakterisiert. (orig./SR)

  15. Identification of Serratia marcescens SE1 and determination of its Herbicide 2,2-dichloropropionate (2,2-DCP Degradation Potential

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    Abel, E.

    2012-01-01

    Full Text Available Aims: The goal of the study is to isolate species of bacteria that capable of utilizing 2,2-dichloropropionic acid (2,2-DCP as sole carbon source from soil sample collected from surrounding lake water located in Universiti Teknologi Malaysia, Skudai, Johor. Methodology and Results: Genomic DNA from bacterium SE1 was extracted and PCR amplification was carried out using universal primers, Fd1 (5’ - AGA GTT TGA TCC TGGCTC AG - 3’ and rP1 (5’- ACG GTC ATA CCT TGT TAC GAC TT - 3’ before sending for sequencing. The 16S rDNA nucleotide sequences were compared with Basic Local Alignment Search Tool nucleotide (BLASTn and further analyzed using phylogenetic tree of Neighbour-Joining method (MEGA 5. Phylogenetic analysis indicated that SE1 strain clearly shared 97% homology to the genus of Serratia marcescens and therefore designated as Serratia marcescens sp. SE1. SE1 exhibited the ability to utilize 2,2-DCP as sole carbon source at 20 mM concentration with cell doubling time of 5 h and maximum chloride ion release of 38 μmolCl-/mL. This result suggests that the dehalogenase enzyme present in the bacteria has high affinity towards the substrate. Based on morphological and partial biochemical characteristics, strain SE1 was a non-motile Gram negative bacterium with red colonies, that gave a catalase positive reaction. Conclusion, significance and impact of study: A better understanding of dehalogenases enzyme produce by this S. marcescens sp. SE1 in general will be useful to be used as bioremediation tools for environmental management. This is the first reported case that Serratia sp. has the ability to degrade halogenated compound.

  16. [Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase].

    Science.gov (United States)

    Firsova, Iu E; Doronina, N V; Trotsenko, Iu A

    2004-01-01

    The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.

  17. Phenol Is the Initial Product Formed during Growth and Degradation of Bromobenzene by Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 via an Early Dehalogenation Step.

    Science.gov (United States)

    Vatsal, Aakanksha A; Zinjarde, Smita S; RaviKumar, Ameeta

    2017-01-01

    Bromobenzene (BrB), a hydrophobic, recalcitrant organic compound, is listed by the environmental protection agencies as an environmental and marine pollutant having hepatotoxic, mutagenic, teratogenic, and carcinogenic effects. The tropical marine yeast Yarrowia lipolytica 3589 was seen to grow aerobically on BrB and displayed a maximum growth rate (μ max ) of 0.04 h -1 . Furthermore, we also observed an increase in cell size and sedimentation velocity for the cells grown on BrB as compared to the glucose grown cells. The cells attached to the hydrophobic bromobenzene droplets through its hydrophobic and acid-base interactions. The BrB (0.5%, 47.6 mM) was utilized by the cells with the release of a corresponding amount of bromide (12.87 mM) and yielded a cell mass of 1.86 g/L after showing 34% degradation in 96 h. Maximum dehalogenase activity of 16.16 U/mL was seen in the cell free supernatant after 24 h of growth. Identification of metabolites formed as a result of BrB degradation, namely, phenol, catechol, cis, cis muconic acid, and carbon dioxide were determined by LC-MS and GC-MS. The initial attack on bromobenzene by Y. lipolytica cells lead to the transient accumulation of phenol as an early intermediate which is being reported for the first time. Degradation of phenol led to catechol which was degraded by the ortho- cleavage pathway forming cis, cis muconic acid and then to Krebs cycle intermediates eventually leading to CO 2 production. The study shows that dehalogenation via an extracellular dehalogenase occurs prior to ring cleavage with phenol as the preliminary degradative compound being produced. The yeast was also able to grow on the degradative products, i.e., phenol and catechol, to varying degrees which would be of potential relevance in the degradation and remediation of xenobiotic environmental bromoaromatic pollutants such as bromobenzene.

  18. Phenol Is the Initial Product Formed during Growth and Degradation of Bromobenzene by Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 via an Early Dehalogenation Step

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    Aakanksha A. Vatsal

    2017-06-01

    Full Text Available Bromobenzene (BrB, a hydrophobic, recalcitrant organic compound, is listed by the environmental protection agencies as an environmental and marine pollutant having hepatotoxic, mutagenic, teratogenic, and carcinogenic effects. The tropical marine yeast Yarrowia lipolytica 3589 was seen to grow aerobically on BrB and displayed a maximum growth rate (μmax of 0.04 h-1. Furthermore, we also observed an increase in cell size and sedimentation velocity for the cells grown on BrB as compared to the glucose grown cells. The cells attached to the hydrophobic bromobenzene droplets through its hydrophobic and acid–base interactions. The BrB (0.5%, 47.6 mM was utilized by the cells with the release of a corresponding amount of bromide (12.87 mM and yielded a cell mass of 1.86 g/L after showing 34% degradation in 96 h. Maximum dehalogenase activity of 16.16 U/mL was seen in the cell free supernatant after 24 h of growth. Identification of metabolites formed as a result of BrB degradation, namely, phenol, catechol, cis, cis muconic acid, and carbon dioxide were determined by LC–MS and GC–MS. The initial attack on bromobenzene by Y. lipolytica cells lead to the transient accumulation of phenol as an early intermediate which is being reported for the first time. Degradation of phenol led to catechol which was degraded by the ortho- cleavage pathway forming cis, cis muconic acid and then to Krebs cycle intermediates eventually leading to CO2 production. The study shows that dehalogenation via an extracellular dehalogenase occurs prior to ring cleavage with phenol as the preliminary degradative compound being produced. The yeast was also able to grow on the degradative products, i.e., phenol and catechol, to varying degrees which would be of potential relevance in the degradation and remediation of xenobiotic environmental bromoaromatic pollutants such as bromobenzene.

  19. Anaerobic degradation of tetrachloroethylene; Anaerober Abbau von Tetrachlorethylen

    Energy Technology Data Exchange (ETDEWEB)

    Diekert, G. [Stuttgart Univ. (Germany). Inst. fuer Mikrobiologie; Scholz-Muramatsu, H. [Stuttgart Univ. (Germany). Inst. fuer Siedlungswasserbau

    1996-12-31

    Dehalospirillum multivorans, a tetrachloroethylene-dechlorinating bacterium, was isolated in activated sludge. This organism is able to grow on a defined medium with hydrogen and tetrachloroethylene (PCE) as its only energy source. The organism was characterised and the physiology of dechlorination was studied. In this process PCE is dechlorinated to cis-1,2-dichloroethene (DCE) via trichloroethene (TCE). A fluidized-bed reactor which reduces PCE to DCE at a high rate (15 nmol/min/mg of protein at 5 {mu}M PCE) was inoculated with the bacterium. Meanwhile a reactor inoculated with D. multivorans and a fully dechlorinating mixed culture has become available which catalyses the complete dechlorination of PCE to ethene at just as high rates. Tetrachloroethene dehalogenase was purified from D. multivorans (unpublished results) and characterised. (orig./SR) [Deutsch] Aus Belebtschlamm wurde ein Tetrachlorethen-dechlorierendes Bakterium, Dehalospirillum multivorans, isoliert. Der Organismus waechst auf definiertem Medium mit Wasserstoff und Tetrachlorethen (PCE) als einziger Energiequelle. Der Organismus wurde charakterisiert und die Physiologie der Dechlorierung wurde untersucht. PCE wird dabei ueber Trichlorethen (TCE) bis zum cis-1,2-Dichlorethen (DCE) dechloriert. Mit diesem Bakterium wurde ein Wirbelschichtreaktor inokuliert, der mit hohen Raten (15 nmol/min/mg Protein bei 5 {mu}M PCE) PCE zu DCE reduziert. Inzwischen steht ein Reaktor zur Verfuegung, der mit D. multivorans und einer voellig dechlorierenden Mischkultur inokuliert wurde und der mit ebenso hohen Raten eine vollstaendige Dechlorierung von PCE bis zum Ethen katalysiert. Aus D. multivorans wurde die Tetrachlorethen-Dehalogenase gereinigt (unveroeffentlichte Ergebnisse) und charakterisiert. (orig./SR)

  20. Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis.

    Directory of Open Access Journals (Sweden)

    Simon Oakley

    Full Text Available Barbiturates potentiate GABA actions at the GABA(A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10-500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

  1. Whole-Genome Sequence Analysis of Antimicrobial Resistance Genes in Streptococcus uberis and Streptococcus dysgalactiae Isolates from Canadian Dairy Herds

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    Julián Reyes Vélez

    2017-05-01

    Full Text Available The objectives of this study are to determine the occurrence of antimicrobial resistance (AMR genes using whole-genome sequence (WGS of Streptococcus uberis (S. uberis and Streptococcus dysgalactiae (S. dysgalactiae isolates, recovered from dairy cows in the Canadian Maritime Provinces. A secondary objective included the exploration of the association between phenotypic AMR and the genomic characteristics (genome size, guanine–cytosine content, and occurrence of unique gene sequences. Initially, 91 isolates were sequenced, and of these isolates, 89 were assembled. Furthermore, 16 isolates were excluded due to larger than expected genomic sizes (>2.3 bp × 1,000 bp. In the final analysis, 73 were used with complete WGS and minimum inhibitory concentration records, which were part of the previous phenotypic AMR study, representing 18 dairy herds from the Maritime region of Canada (1. A total of 23 unique AMR gene sequences were found in the bacterial genomes, with a mean number of 8.1 (minimum: 5; maximum: 13 per genome. Overall, there were 10 AMR genes [ANT(6, TEM-127, TEM-163, TEM-89, TEM-95, Linb, Lnub, Ermb, Ermc, and TetS] present only in S. uberis genomes and 2 genes unique (EF-TU and TEM-71 to the S. dysgalactiae genomes; 11 AMR genes [APH(3′, TEM-1, TEM-136, TEM-157, TEM-47, TetM, bl2b, gyrA, parE, phoP, and rpoB] were found in both bacterial species. Two-way tabulations showed association between the phenotypic susceptibility to lincosamides and the presence of linB (P = 0.002 and lnuB (P < 0.001 genes and the between the presence of tetM (P = 0.015 and tetS (P = 0.064 genes and phenotypic resistance to tetracyclines only for the S. uberis isolates. The logistic model showed that the odds of resistance (to any of the phenotypically tested antimicrobials was 4.35 times higher when there were >11 AMR genes present in the genome, compared with <7 AMR genes (P < 0.001. The odds of resistance was lower for S

  2. III-Nitrides growth and AlGaN/GaN heterostructures on ferroelectric materials

    International Nuclear Information System (INIS)

    Lee, Kyoung-Keun; Namkoong, Gon; Madison, Shannon M.; Ralph, Stephen E.; Doolittle, W. Alan; Losurdo, Maria; Bruno, Giovanni; Cho, Hyung Koun

    2007-01-01

    The growth of III-nitrides on the ferroelectric materials lithium niobate (LN) and lithium tantalate (LT) via molecular beam epitaxy (MBE) using rf plasma source has been investigated. We have found that gallium nitride (GaN) epitaxial layers have a crystalline relationship with lithium niobate (tantalate) as follows: (0 0 0 1) GaN || (0 0 0 1) LN (LT) with [10-10] GaN || [11-20] LN (LT). The surface stability of LN and LT substrates has been monitored by in situ spectroscopic ellipsometry in the vacuum chamber. Three different temperature zones have been discerned; surface degas and loss of OH group (100-350 deg. C); surface segregation/accumulation of Li and O-species (400-700 deg. C); surface evaporation of O-species and Li desorption (over 750 deg. C). However, LT shows only surface degassing in the range of 100-800 deg. C. Therefore, congruent LN substrates were chemically unstable at the growth temperature of 550-650 deg. C, and therefore developed an additional phase of Li-deficient lithium niobate (LiNb 3 O 8 ) along with lithium niobate (LiNbO 3 ), confirmed by X-ray diffraction. On the other hand, LT showed better chemical stability at these temperatures, with no additional phase development. The structural quality of GaN epitaxial layers has shown slight improvement on LT substrates over LN substrates, according to X-ray diffraction. Herein, we demonstrate AlGaN/GaN heterostructure devices on ferroelectric materials that will allow future development of multifunctional electrical and optical applications

  3. Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection.

    Science.gov (United States)

    de Moura, Tatiana R; Oliveira, Fabiano; Carneiro, Marcia W; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M C; Valenzuela, Jesus G; de Oliveira, Camila I

    2013-01-01

    Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

  4. The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12- and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study.

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    Sandro Roselli

    Full Text Available Chloromethane (CH3Cl is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD, as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2. In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex, conversion of

  5. Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere.

    Science.gov (United States)

    Fullerton, Heather; Moyer, Craig L

    2016-05-15

    Chloroflexi small-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylum Chloroflexi is represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurface Chloroflexi Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurface Chloroflexi single amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nine Chloroflexi SAGs (seven of which are from the order Anaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of the Chloroflexi SAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the seven Anaerolineales SAGs contains a distinct reductive dehalogenase homologous (rdhA) gene. Through the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, the Chloroflexi These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examined Chloroflexi SAGs, our genomes (several are from the order Anaerolineales) were recovered from a hydrothermally driven system and therefore provide a unique window into

  6. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway.

    Science.gov (United States)

    Dvorak, Pavel; Chrast, Lukas; Nikel, Pablo I; Fedr, Radek; Soucek, Karel; Sedlackova, Miroslava; Chaloupkova, Radka; de Lorenzo, Víctor; Prokop, Zbynek; Damborsky, Jiri

    2015-12-21

    Heterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer's chemical properties. IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of P lac , dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation

  7. Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides.

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    Lygia T Budnik

    Full Text Available There is a need for a panel of suitable biomarkers for detection of environmental chemical exposure leading to the initiation or progression of degenerative diseases or potentially, to cancer. As the peripheral blood may contain increased levels of circulating cell-free DNA in diseased individuals, we aimed to evaluate this DNA as effect biomarker recognizing vulnerability after exposure to environmental chemicals. We recruited 164 individuals presumably exposed to halo-alkane-based pesticides. Exposure evaluation was based on human biomonitoring analysis; as biomarker of exposure parent halo-methanes, -ethanes and their metabolites, as well as the hemoglobin-adducts methyl valine and hydroxyl ethyl valine in blood were used, complemented by expert evaluation of exposure and clinical intoxication symptoms as well as a questionnaire. Assessment showed exposures to halo alkanes in the concentration range being higher than non-cancer reference doses (RfD but (mostly lower than the occupational exposure limits. We quantified circulating DNA in serum from 86 individuals with confirmed exposure to off-gassing halo-alkane pesticides (in storage facilities or in home environment and 30 non-exposed controls, and found that exposure was significantly associated with elevated serum levels of circulating mitochondrial DNA (in size of 79 bp, mtDNA-79, p = 0.0001. The decreased integrity of mtDNA (mtDNA-230/mtDNA-79 in exposed individuals implicates apoptotic processes (p = 0.015. The relative amounts of mtDNA-79 in serum were positively associated with the lag-time after intoxication to these chemicals (r = 0.99, p<0.0001. Several months of post-exposure the specificity of this biomarker increased from 30% to 97% in patients with intoxication symptoms. Our findings indicate that mitochondrial DNA has a potential to serve as a biomarker recognizing vulnerable risk groups after exposure to toxic/carcinogenic chemicals.

  8. Structural and molecular basis for resistance to aminoglycoside antibiotics by the adenylyltransferase ANT(2″)-Ia.

    Science.gov (United States)

    Cox, Georgina; Stogios, Peter J; Savchenko, Alexei; Wright, Gerard D

    2015-01-06

    The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE  : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular

  9. Lithiotantite, ideally LiTa3O8

    Directory of Open Access Journals (Sweden)

    Aba C. Persiano

    2012-05-01

    Full Text Available Lithiotantite (lithium tritantalum octaoxide and lithiowodginite are natural dimorphs of LiTa3O8, corresponding to the laboratory-synthesized L-LiTa3O8 (low-temperature form and M-LiTa3O8 (intermediate-temperature form phases, respectively. Based on single-crystal X-ray diffraction data, this study presents the first structure determination of lithiotantite from a new locality, the Murundu mine, Jenipapo District, Itinga, Minas Gerais, Brazil. Lithiotantite is isotypic with LiNb3O8 and its structure is composed of a slightly distorted hexagonal close-packed array of O atoms stacked in the [-101] direction, with the metal atoms occupying half of the octahedral sites. There are four symmetrically non-equivalent cation sites, with three of them occupied mainly by (Ta5+ + Nb5+ and one by Li+. The four distinct octahedra share edges, forming two types of zigzag chains (A and B extending along the b axis. The A chains are built exclusively of (Ta,NbO6 octahedra (M1 and M2, whereas the B chains consist of alternating (Ta,NbO6 and LiO6 octahedra (M3 and M4, respectively. The average M1—O, M2—O, M3—O and M4—O bond lengths are 2.011, 2.004, 1.984, and 2.188 Å, respectively. Among the four octahedra, M3 is the least distorted and M4 the most. The refined Ta contents at the M1, M2 and M3 sites are 0.641 (2, 0.665 (2, and 0.874 (2, respectively, indicating a strong preference of Ta5+ for M3 in the B chain. The refined composition of the crystal investigated is Li0.96Mn0.03Na0.01Nb0.82Ta2.18O8.

  10. Molecular identification and biodegradation of 3-chloropropionic acid (3CP by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

    Directory of Open Access Journals (Sweden)

    Parvizpour, S.

    2013-01-01

    Full Text Available Aims: This study was carried out to further characterize fungal species that could degrade 3-chloropropionic acid (3CPas sole source of carbon and energy. Methodology and Results: Both fungi were able to grow on 3CP after 10 days on solid minimal media. Based on sequencing of its segment of 18S rRNA these isolates were identified as Mucor sp. SP1 and Trichoderma sp. SP2. The isolated strains were not able to grow on media plates containing 10 mM of 2,2-dichloropropionate (2,2DCP as sole source of carbon. 3CP degradation was observed in liquid minimal medium containing 10 mM 3CP after 18 days cultureperiod. The chloride ion released was detected in both growth medium containing Mucor sp. SP1 and Trichoderma sp. SP2. At least 80% of 10 mM 3CP was utilized in the growth medium. Conclusion, significance and impact of study: Dehalogenase enzyme that can degrade α-chloro-substituted haloalkanoic acids for example 2,2DCP is well studied up to protein crystallization. Very few reports on the degradationof β-chloro-substituted haloalkanoic acids such as 3CP and none from fungi. This study is considered important because it can be compared to that of well-documented α-chloro-substituted haloalkanoic acids degradation. This is the first study to indicate fungal growth on 3CP as sole carbon and energy sources.

  11. Sulfobacillus thermosulfidooxidans strain Cutipay enhances chalcopyrite bioleaching under moderate thermophilic conditions in the presence of chloride ion.

    Science.gov (United States)

    Bobadilla-Fazzini, Roberto A; Cortés, Maria Paz; Maass, Alejandro; Parada, Pilar

    2014-12-01

    Currently more than 90% of the world's copper is obtained through sulfide mineral processing. Among the copper sulfides, chalcopyrite is the most abundant and therefore economically relevant. However, primary copper sulfide bioleaching is restricted due to high ionic strength raffinate solutions and particularly chloride coming from the dissolution of ores. In this work we describe the chalcopyrite bioleaching capacity of Sulfobacillus thermosulfidooxidans strain Cutipay (DSM 27601) previously described at the genomic level (Travisany et al. (2012) Draft genome sequence of the Sulfobacillus thermosulfidooxidans Cutipay strain, an indigenous bacterium isolated from a naturally extreme mining environment in Northern Chile. J Bacteriol 194:6327-6328). Bioleaching assays with the mixotrophic strain Cutipay showed a strong increase in copper recovery from chalcopyrite concentrate at 50°C in the presence of chloride ion, a relevant inhibitory element present in copper bioleaching processes. Compared to the abiotic control and a test with Sulfobacillus acidophilus DSM 10332, strain Cutipay showed an increase of 42 and 69% in copper recovery, respectively, demonstrating its high potential for chalcopyrite bioleaching. Moreover, a genomic comparison highlights the presence of the 2-Haloacid dehalogenase predicted-protein related to a potential new mechanism of chloride resistance in acidophiles. This novel and industrially applicable strain is under patent application CL 2013-03335.

  12. Fast and effective transformation of toxaphene by superreduced vitamin B12 and dicyanocobalamin

    Energy Technology Data Exchange (ETDEWEB)

    Recke, R. von der; Gaul, S. [Inst. of Food Chemistry, Univ. of Hohenheim, Stuttgart (Germany); Ruppe, S.; Vetter, W. [Inst. of Food and Nutrition, Friedrich-Schiller-Univ. Jena (Germany); Neumann, A. [Dept. of Technical Biology, Univ. of Karlsruhe (Germany)

    2004-09-15

    The chloropesticide toxaphene (Camphechlor, Melipax) has been used in high quantities since 1945. Toxaphene has been classified as a persistent organohalogen pollutant (POP) and belongs to the ''dirty dozen''. Due to its heavy use in the 1960s and 1970s, several environmental compartments were significantly contaminated with toxaphene. For instance, concentrations in polluted areas could exceed 1 mg/kg sediment. Recently, it was shown that cultures of the isolated bacterium Dehalospirillum multivorans transformed toxaphene in a similar way as anaerobic sediment and soil samples. The potential of D. multivorans for the anaerobic transformation of organohalogens was previously demonstrated for chloroethenes (PCE and TCE) 6. The reactive dehalogenase of D. multivorans contains a corrinoide co-factor. In this study two corrinoids cyanocobalamin (vitamin B12, CCA) and dicyanocobinamide (DCC) were used in their superreduced forms (i. e. those having the central atom Co in the oxidation state +I). Interestingly, a very fast transformation of toxaphene was observed with superreduced DCC. Unfortunately, the transformation occurred so fast that no transformation products could be identified. For this reason, we performed further experiments with the less reactive superreduced vitamin B12 (CCA{sub s}). CCA{sub s} has previously been used for the transformation of PCBs, hexachlorobenzene, chloroform, and chloroethanes.

  13. YbiV from E. coli K12 is a HAD phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Anne; Lee, Seok-Yong; McCullagh, Emma; Silversmith, Ruth E.; Wemmer, David E.

    2004-03-16

    The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

  14. A Stevedore's protein knot.

    Directory of Open Access Journals (Sweden)

    Daniel Bölinger

    2010-04-01

    Full Text Available Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered alpha-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip.

  15. Identifying and sequencing a Mycobacterium sp. strain F4 as a potential bioremediation agent for quinclorac.

    Science.gov (United States)

    Li, Yingying; Chen, Wu; Wang, Yunsheng; Luo, Kun; Li, Yue; Bai, Lianyang; Luo, Feng

    2017-01-01

    Quinclorac is a widely used herbicide in rice filed. Unfortunately, quinclorac residues are phytotoxic to many crops/vegetables. The degradation of quinclorac in nature is very slow. On the other hand, degradation of quinclorac using bacteria can be an effective and efficient method to reduce its contamination. In this study, we isolated a quinclorac bioremediation bacterium strain F4 from quinclorac contaminated soils. Based on morphological characteristics and 16S rRNA gene sequence analysis, we identified strain F4 as Mycobacterium sp. We investigated the effects of temperature, pH, inoculation size and initial quinclorac concentration on growth and degrading efficiency of F4 and determined the optimal quinclorac degrading condition of F4. Under optimal degrading conditions, F4 degraded 97.38% of quinclorac from an initial concentration of 50 mg/L in seven days. Our indoor pot experiment demonstrated that the degradation products were non-phytotoxic to tobacco. After analyzing the quinclorac degradation products of F4, we proposed that F4 could employ two pathways to degrade quinclorac: one is through methylation, the other is through dechlorination. Furthermore, we reconstructed the whole genome of F4 through single molecular sequencing and de novo assembly. We identified 77 methyltransferases and eight dehalogenases in the F4 genome to support our hypothesized degradation path.

  16. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid

    International Nuclear Information System (INIS)

    Miranda-Rojas, Sebastián; Toro-Labbé, Alejandro

    2015-01-01

    Fluoroacetate is a toxic compound whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degradation through a two step process initiated by an S N 2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on density functional theory calculations of the S N 2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the S N 2 reaction. In this way, a site-induced partial protonation acts as a chemical switch in a key process that determines the output of the reaction

  17. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

    Science.gov (United States)

    Renata, Hans; Wang, Z. Jane

    2015-01-01

    High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

  18. Reductive dehalogenation activity of indigenous microorganism in sediments of the Hackensack River, New Jersey.

    Science.gov (United States)

    Sohn, Seo Yean; Häggblom, Max M

    2016-07-01

    Organohalogen pollutants are of concern in many river and estuarine environments, such as the New York-New Jersey Harbor estuary and its tributaries. The Hackensack River is contaminated with various metals, hydrocarbons and halogenated organics, including polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins. In order to examine the potential for microbial reductive dechlorination by indigenous microorganisms, sediment samples were collected from five different estuarine locations along the Hackensack River. Hexachlorobenzene (HCB), hexabromobenzene (HBB), and pentachloroaniline (PCA) were selected as model organohalogen pollutants to assess anaerobic dehalogenating potential. Dechlorinating activity of HCB and PCA was observed in sediment microcosms for all sampling sites. HCB was dechlorinated via pentachlorobenzene (PeCB) and trichlorobenzene (TriCB) to dichlorobenzene (DCB). PCA was dechlorinated via tetrachloroaniline (TeCA), trichloroanilines (TriCA), and dichloroanilines (DCA) to monochloroaniline (MCA). No HBB debromination was observed over 12 months of incubation. However, with HCB as a co-substrate slow HBB debromination was observed with production of tetrabromobenzene (TeBB) and tribromobenzene (TriBB). Chloroflexi specific 16S rRNA gene PCR-DGGE followed by sequence analysis detected Dehalococcoides species in sediments of the freshwater location, but not in the estuarine site. Analysis targeting 12 putative reductive dehalogenase (rdh) genes showed that these were enriched concomitant with HCB or PCA dechlorination in freshwater sediment microcosms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Distribution of dehalogenation activity in subseafloor sediments of the Nankai Trough subduction zone.

    Science.gov (United States)

    Futagami, Taiki; Morono, Yuki; Terada, Takeshi; Kaksonen, Anna H; Inagaki, Fumio

    2013-04-19

    Halogenated organic matter buried in marine subsurface sediment may serve as a source of electron acceptors for anaerobic respiration of subseafloor microbes. Detection of a diverse array of reductive dehalogenase-homologous (rdhA) genes suggests that subseafloor organohalide-respiring microbial communities may play significant ecological roles in the biogeochemical carbon and halogen cycle in the subseafloor biosphere. We report here the spatial distribution of dehalogenation activity in the Nankai Trough plate-subduction zone of the northwest Pacific off the Kii Peninsula of Japan. Incubation experiments with slurries of sediment collected at various depths and locations showed that degradation of several organohalides tested only occurred in the shallow sedimentary basin, down to 4.7 metres below the seafloor, despite detection of rdhA in the deeper sediments. We studied the phylogenetic diversity of the metabolically active microbes in positive enrichment cultures by extracting RNA, and found that Desulfuromonadales bacteria predominate. In addition, for the isolation of genes involved in the dehalogenation reaction, we performed a substrate-induced gene expression screening on DNA extracted from the enrichment cultures. Diverse DNA fragments were obtained and some of them showed best BLAST hit to known organohalide respirers such as Dehalococcoides, whereas no functionally known dehalogenation-related genes such as rdhA were found, indicating the need to improve the molecular approach to assess functional genes for organohalide respiration.

  20. Proteomic data set of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates

    Directory of Open Access Journals (Sweden)

    Tobias Goris

    2016-09-01

    Full Text Available Sulfurospirillum multivorans is a free-living, physiologically versatile Epsilonproteobacterium able to couple the reductive dehalogenation of chlorinated and brominated ethenes to growth (organohalide respiration. We present proteomic data of S. multivorans grown with different electron donors (formate or pyruvate and electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]. To obtain information on the cellular localization of proteins, membrane extracts and soluble fractions were separated before data collection from both fractions. The proteome analysis of S. multivorans was performed by mass spectrometry (nanoLC-MS/MS. Raw data have been deposited at ProteomeXchange, “ProteomeXchange provides globally coordinated proteomics data submission and dissemination” [1], via the PRIDE partner repository with the dataset identifier PRIDE: PXD004011. The data might support further research in organohalide respiration and in the general metabolism of free-living Epsilonproteobacteria. The dataset is associated with a previously published study “Proteomics of the organohalide-respiring Epsilonproteobacterium S. multivorans adapted to tetrachloroethene and other energy substrates” [2]. Keywords: Anaerobic respiration, Epsilonproteobacteria, Nitrate respiration, Reductive dechlorination, Reductive dehalogenase

  1. Reductive tetrachloroethene dehalogenation in the presence of oxygen by Sulfurospirillum multivorans: physiological studies and proteome analysis.

    Science.gov (United States)

    Gadkari, Jennifer; Goris, Tobias; Schiffmann, Christian L; Rubick, Raffael; Adrian, Lorenz; Schubert, Torsten; Diekert, Gabriele

    2018-01-01

    Reductive dehalogenation of organohalides is carried out by organohalide-respiring bacteria (OHRB) in anoxic environments. The tetrachloroethene (PCE)-respiring Epsilonproteobacterium Sulfurospirillum multivorans is one of few OHRB able to respire oxygen. Therefore, we investigated the organism's capacity to dehalogenate PCE in the presence of oxygen, which would broaden the applicability to use S. multivorans, unlike other commonly oxygen-sensitive OHRB, for bioremediation, e.g. at oxic/anoxic interphases. Additionally, this has an impact on our understanding of the global halogen cycle. Sulfurospirillum multivorans performs dehalogenation of PCE to cis-1,2-dichloroethene at oxygen concentrations below 0.19 mg/L. The redox potential of the medium electrochemically adjusted up to +400 mV had no influence on reductive dehalogenation by S. multivorans in our experiments, suggesting that higher levels of oxygen impair PCE dechlorination by inhibiting or inactivating involved enzymes. The PCE reductive dehalogenase remained active in cell extracts of S. multivorans exposed to 0.37 mg/L oxygen for more than 96 h. Analysis of the proteome revealed that superoxide reductase and cytochrome peroxidase amounts increased with 5% oxygen in the gas phase, while the response to atmospheric oxygen concentrations involved catalase and hydrogen peroxide reductase. Taken together, our results demonstrate that reductive dehalogenation by OHRB is not limited to anoxic conditions. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Binding of anions in triply interlocked coordination catenanes and dynamic allostery for dehalogenation reactions† †Electronic supplementary information (ESI) available: Characterization data and additional tables and figures. CCDC 1515722 and 1515723. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7sc04070a

    Science.gov (United States)

    Yang, Linlin; Jing, Xu; An, Bowen; Yang, Yang

    2017-01-01

    By synergistic combination of multicomponent self-assembly and template-directed approaches, triply interlocked metal organic catenanes that consist of two isolated chirally identical tetrahedrons were constructed and stabilized as thermodynamic minima. In the presence of suitable template anions, the structural conversion from the isolated tetrahedral conformers into locked catenanes occurred via the cleavage of an intrinsically reversible coordination bond in each of the tetrahedrons, followed by the reengineering and interlocking of two fragments with the regeneration of the broken coordination bonds. The presence of several kinds of individual pocket that were attributed to the triply interlocked patterns enabled the possibility of encapsulating different anions, allowing the dynamic allostery between the unlocked/locked conformers to promote the dehalogenation reaction of 3-bromo-cyclohexene efficiently, as with the use of dehalogenase enzymes. The interlocked structures could be unlocked into two individual tetrahedrons through removal of the well-matched anion templates. The stability and reversibility of the locked/unlocked structures were further confirmed by the catching/releasing process that accompanied emission switching, providing opportunities for the system to be a dynamic molecular logic system. PMID:29675152

  3. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid

    Science.gov (United States)

    Miranda-Rojas, Sebastián; Toro-Labbé, Alejandro

    2015-05-01

    Fluoroacetate is a toxic compound whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degradation through a two step process initiated by an SN2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on density functional theory calculations of the SN2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the SN2 reaction. In this way, a site-induced partial protonation acts as a chemical switch in a key process that determines the output of the reaction.

  4. Reductive dehalogenation of 3,5-dibromo-4-hydroxybenzoate by an aerobic strain of Delftia sp. EOB-17.

    Science.gov (United States)

    Chen, Kai; Jian, Shanshan; Huang, Linglong; Ruan, Zhepu; Li, Shunpeng; Jiang, Jiandong

    2015-12-01

    To confirm the reductive dehalogenation ability of the aerobic strain of Delftia sp. EOB-17, finding more evidences to support the hypothesis that reductive dehalogenation may occur extensively in aerobic bacteria. Delftia sp. EOB-17, isolated from terrestrial soil contaminated with halogenated aromatic compounds, completely degraded 0.2 mM DBHB in 28 h and released two equivalents of bromides under aerobic conditions in the presence of sodium succinate. LC-MS analysis revealed that DBHB was transformed to 4-hydroxybenzoate via 3-bromo-4-hydroxybenzoate by successive reductive dehalogenation. Highly conserved DBHB-degrading genes, including reductive dehalogenase gene (bhbA3) and the extra-cytoplasmic binding receptor gene (bhbB3), were also found in strain EOB-17 by genome sequencing. The optimal temperature and pH for DBHB reductive dehalogenation activity are 30 °C and 8, respectively, and 0.1 mM Cd(2+), Cu(2+), Hg(2+) and Zn(2+) strongly inhibited dehalogenation activity. The aerobic strain of Delftia sp. EOB-17 was confirmed to reductively dehalogenate DBHB under aerobic conditions, providing another evidence to support the hypothesis that reductive dehalogenation occurs extensively in aerobic bacteria.

  5. Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation

    Science.gov (United States)

    Townsend, G. T.; Suflita, J. M.

    1996-01-01

    We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates. PMID:16535377

  6. Mechanistic insights into the dehalogenation reaction of fluoroacetate/fluoroacetic acid

    Energy Technology Data Exchange (ETDEWEB)

    Miranda-Rojas, Sebastián, E-mail: sebastian.miranda@unab.cl [Chemical Processes and Catalysis (CPC), Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andres Bello, Avenida República 275, Santiago (Chile); Toro-Labbé, Alejandro [Laboratorio de Química Teórica Computacional (QTC), Facultad de Química, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Macul, Santiago (Chile)

    2015-05-21

    Fluoroacetate is a toxic compound whose environmental accumulation may represent an important contamination problem, its elimination is therefore a challenging issue. Fluoroacetate dehalogenase catalyzes its degradation through a two step process initiated by an S{sub N}2 reaction in which the aspartate residue performs a nucleophilic attack on the carbon bonded to the fluorine; the second step is hydrolysis that releases the product as glycolate. In this paper, we present a study based on density functional theory calculations of the S{sub N}2 initiation reaction modeled through the interaction between the substrate and the propionate anion as the nucleophile. Results are analyzed within the framework of the reaction force and using the reaction electronic flux to identify and characterize the electronic activity that drives the reaction. Our results reveal that the selective protonation of the substrate catalyzes the reaction by decreasing the resistance of the structural and electronic reorganization needed to reach the transition state. Finally, the reaction energy is modulated by the degree of stabilization of the fluoride anion formed after the S{sub N}2 reaction. In this way, a site-induced partial protonation acts as a chemical switch in a key process that determines the output of the reaction.

  7. Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

    Science.gov (United States)

    Kunze, Cindy; Bommer, Martin; Hagen, Wilfred R; Uksa, Marie; Dobbek, Holger; Schubert, Torsten; Diekert, Gabriele

    2017-07-03

    The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B 12 -dependent biochemistry and represents an effective mode of RDase catalysis.

  8. Application of bioinformatics tools and databases in microbial dehalogenation research (a review).

    Science.gov (United States)

    Satpathy, R; Konkimalla, V B; Ratha, J

    2015-01-01

    Microbial dehalogenation is a biochemical process in which the halogenated substances are catalyzed enzymatically in to their non-halogenated form. The microorganisms have a wide range of organohalogen degradation ability both explicit and non-specific in nature. Most of these halogenated organic compounds being pollutants need to be remediated; therefore, the current approaches are to explore the potential of microbes at a molecular level for effective biodegradation of these substances. Several microorganisms with dehalogenation activity have been identified and characterized. In this aspect, the bioinformatics plays a key role to gain deeper knowledge in this field of dehalogenation. To facilitate the data mining, many tools have been developed to annotate these data from databases. Therefore, with the discovery of a microorganism one can predict a gene/protein, sequence analysis, can perform structural modelling, metabolic pathway analysis, biodegradation study and so on. This review highlights various methods of bioinformatics approach that describes the application of various databases and specific tools in the microbial dehalogenation fields with special focus on dehalogenase enzymes. Attempts have also been made to decipher some recent applications of in silico modeling methods that comprise of gene finding, protein modelling, Quantitative Structure Biodegradibility Relationship (QSBR) study and reconstruction of metabolic pathways employed in dehalogenation research area.

  9. Binding of anions in triply interlocked coordination catenanes and dynamic allostery for dehalogenation reactions.

    Science.gov (United States)

    Yang, Linlin; Jing, Xu; An, Bowen; He, Cheng; Yang, Yang; Duan, Chunying

    2018-01-28

    By synergistic combination of multicomponent self-assembly and template-directed approaches, triply interlocked metal organic catenanes that consist of two isolated chirally identical tetrahedrons were constructed and stabilized as thermodynamic minima. In the presence of suitable template anions, the structural conversion from the isolated tetrahedral conformers into locked catenanes occurred via the cleavage of an intrinsically reversible coordination bond in each of the tetrahedrons, followed by the reengineering and interlocking of two fragments with the regeneration of the broken coordination bonds. The presence of several kinds of individual pocket that were attributed to the triply interlocked patterns enabled the possibility of encapsulating different anions, allowing the dynamic allostery between the unlocked/locked conformers to promote the dehalogenation reaction of 3-bromo-cyclohexene efficiently, as with the use of dehalogenase enzymes. The interlocked structures could be unlocked into two individual tetrahedrons through removal of the well-matched anion templates. The stability and reversibility of the locked/unlocked structures were further confirmed by the catching/releasing process that accompanied emission switching, providing opportunities for the system to be a dynamic molecular logic system.

  10. Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence.

    Science.gov (United States)

    Fang, Xiangling; Barbetti, Martin J

    2014-08-28

    This study was conducted to define differences in Fusarium oxysporum f. sp. fragariae (Fof) isolates with different virulence efficiency to strawberry at the proteome level, in combination with their differences in mycelial growth, conidial production and germination. Comparative proteome analyses revealed substantial differences in mycelial proteomes between Fof isolates, where the 54 differentially accumulated protein spots were consistently over-accumulated or exclusively in the highly virulent isolate. These protein spots were identified through MALDI-TOF/TOF mass spectrometry analyses, and the identified proteins were mainly related to primary and protein metabolism, antioxidation, electron transport, cell cycle and transcription based on their putative functions. Proteins of great potential as Fof virulence factors were those involved in ubiquitin/proteasome-mediated protein degradation and reactive oxygen species detoxification; the hydrolysis-related protein haloacid dehalogenase superfamily hydrolase; 3,4-dihydroxy-2-butanone 4-phosphate synthase associated with riboflavin biosynthesis; and those exclusive to the highly virulent isolate. In addition, post-translational modifications may also make an important contribution to Fof virulence. F. oxysporum f. sp. fragariae (Fof), the causal agent of Fusarium wilt in strawberry, is a serious threat to commercial strawberry production worldwide. However, factors and mechanisms contributing to Fof virulence remained unknown. This study provides knowledge of the molecular basis for the differential expression of virulence in Fof, allowing new possibilities towards developing alternative and more effective strategies to manage Fusarium wilt. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Biodegradation of 2,4'-dichlorobiphenyl, a congener of polychlorinated biphenyl, by Pseudomonas isolates GSa and GSb.

    Science.gov (United States)

    Gayathri, D; Shobha, K J

    2015-08-01

    Bioegradation of 2,4'-dichlorobiphenyl (2,4 CB), by two isolates of Pseudomonas (GSa and GSb) was compared using GC-MS. Transformer oil polluted soil was used for the isolation of 2,4 CB degrading bacteria. GC-MS analysis of the solvent extracts obtained from Pseudomonas sp. GSa spent culture indicated the presence of Phenol 2,6-bis (1,1-dimethyl)-4-methyl (C15H24O). Further, the enzyme analysis of the cell free extracts showed the presence of 2,4'-dichlorobiphenyl dehalogenase (CBD), 2,4'-dichlorobiphenyl-NADPH-oxido-reductase (2,4 CBOR) and 2,3-dihydroxybiphenyl-NADPH-oxido-reductase (2,3 DHOR) with specific activity of 6.00, 0.4 and 0.22 pmol/min/mg of protein, suggesting that dechlorination as an important step during 2,4 CB catabolism. Further, the cell free extract of GSb showed only 2,4'-dichlorobiphenyl-NADPH-oxido-reductase (2,4 CBOR) and 2,3-dihydroxybiphenyl-NADPH-oxido-reductase (2,3 DHOR), with specific activity of 0.3 and 0.213 μmol/min/mg of protein, suggesting attack on non-chlorinated aromatic ring of 2,4 CB, releasing chlorinated intermediates which are toxic to the environment. Although, both the isolated bacteria (GSa and GSb) belong to Pseudomonas spp., they exhibited different metabolic potential.

  12. Electron transport chains in organohalide-respiring bacteria and bioremediation implications.

    Science.gov (United States)

    Wang, Shanquan; Qiu, Lan; Liu, Xiaowei; Xu, Guofang; Siegert, Michael; Lu, Qihong; Juneau, Philippe; Yu, Ling; Liang, Dawei; He, Zhili; Qiu, Rongliang

    2018-04-06

    In situ remediation employing organohalide-respiring bacteria represents a promising solution for cleanup of persistent organohalide pollutants. The organohalide-respiring bacteria conserve energy by utilizing H 2 or organic compounds as electron donors and organohalides as electron acceptors. Reductive dehalogenase (RDase), a terminal reductase of the electron transport chain in organohalide-respiring bacteria, is the key enzyme that catalyzes halogen removal. Accumulating experimental evidence thus far suggests that there are distinct models for respiratory electron transfer in organohalide-respirers of different lineages, e.g., Dehalococcoides, Dehalobacter, Desulfitobacterium and Sulfurospirillum. In this review, to connect the knowledge in organohalide-respiratory electron transport chains to bioremediation applications, we first comprehensively review molecular components and their organization, together with energetics of the organohalide-respiratory electron transport chains, as well as recent elucidation of intramolecular electron shuttling and halogen elimination mechanisms of RDases. We then highlight the implications of organohalide-respiratory electron transport chains in stimulated bioremediation. In addition, major challenges and further developments toward understanding the organohalide-respiratory electron transport chains and their bioremediation applications are identified and discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

    Science.gov (United States)

    Park, Yeonhee; Han, Gil-Soo; Carman, George M

    2017-12-01

    PAH1 -encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions ( e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. The impact of bioaugmentation on dechlorination kinetics and on microbial dechlorinating communities in subsurface clay till

    International Nuclear Information System (INIS)

    Bælum, Jacob; Scheutz, Charlotte; Chambon, Julie C.; Jensen, Christine Mosegaard; Brochmann, Rikke P.; Dennis, Philip; Laier, Troels; Broholm, Mette M.; Bjerg, Poul L.; Binning, Philip J.; Jacobsen, Carsten S.

    2014-01-01

    A molecular study on how the abundance of the dechlorinating culture KB-1 affects dechlorination rates in clay till is presented. DNA extracts showed changes in abundance of specific dechlorinators as well as their functional genes. Independently of the KB-1 added, the microbial dechlorinator abundance increased to the same level in all treatments. In the non-bioaugmented microcosms the reductive dehalogenase gene bvcA increased in abundance, but when KB-1 was added the related vcrA gene increased while bvcA genes did not increase. Modeling showed higher vinyl-chloride dechlorination rates and shorter time for complete dechlorination to ethene with higher initial concentration of KB-1 culture, while cis-dichloroethene dechlorination rates were not affected by KB-1 concentrations. This study provides high resolution abundance profiles of Dehalococcoides spp. (DHC) and functional genes, highlights the ecological behavior of KB-1 in clay till, and reinforces the importance of using multiple functional genes as biomarkers for reductive dechlorination. -- Highlights: • vcrA gene is not always linked to reductive dechlorination potential. • High concentrations of KB-1 stimulate vinyl-chloride degradation. • Vinyl-chloride degradation in non-bioaugmented aquifer is linked to bvcA gene. -- vcrA gene biomarker for reductive dechlorination must be supplemented by bvcA and KB-1 had a positive effect on vinyl-chloride dechlorination compared to dichloroethene dechlorination

  15. Identifying and sequencing a Mycobacterium sp. strain F4 as a potential bioremediation agent for quinclorac.

    Directory of Open Access Journals (Sweden)

    Yingying Li

    Full Text Available Quinclorac is a widely used herbicide in rice filed. Unfortunately, quinclorac residues are phytotoxic to many crops/vegetables. The degradation of quinclorac in nature is very slow. On the other hand, degradation of quinclorac using bacteria can be an effective and efficient method to reduce its contamination. In this study, we isolated a quinclorac bioremediation bacterium strain F4 from quinclorac contaminated soils. Based on morphological characteristics and 16S rRNA gene sequence analysis, we identified strain F4 as Mycobacterium sp. We investigated the effects of temperature, pH, inoculation size and initial quinclorac concentration on growth and degrading efficiency of F4 and determined the optimal quinclorac degrading condition of F4. Under optimal degrading conditions, F4 degraded 97.38% of quinclorac from an initial concentration of 50 mg/L in seven days. Our indoor pot experiment demonstrated that the degradation products were non-phytotoxic to tobacco. After analyzing the quinclorac degradation products of F4, we proposed that F4 could employ two pathways to degrade quinclorac: one is through methylation, the other is through dechlorination. Furthermore, we reconstructed the whole genome of F4 through single molecular sequencing and de novo assembly. We identified 77 methyltransferases and eight dehalogenases in the F4 genome to support our hypothesized degradation path.

  16. Inherited hypothyroidism.

    Science.gov (United States)

    Jackson, I M

    1976-03-01

    Familial hypothyroidism results from both thyroidal and extrathyroidal dysfunction. Specific intrathyroidal abnormalities in thyroid hormone synthesis causing goitrous hypothyroidism are iodide trap defect, organification defect, "coupling" defect, iodoprotein defect, and dehalogenase defect. The diagnostic studies for each are outlined utilizing radioiodine(131I) studies. Other causes of cretinism include failure of the thyroid gland to respond to TSH and lack of pituitary TSH (or hypothalamic TRH). The syndrome of peripheral resistance to thyroid hormone is discussed. The diagnosis of inherited hypothyrodism rests on an adequate family history and measurement of both T4 and TSH levels which can be determined in cord blood or peripheral blood from the infant. The importance of early treatment of hypothyroidism in the neonatal period to prevent brain damage is emphasized. The rec:nt discovery of the importance of reverse T3 (RT3) in fetal thyroid metabolism is described, and the possibility of amniocentesis as an aid in prenatal diagnosis is considered. The place of intrauterine administration of thyroid hormone to the fetus at risk from hypothyroidism is uncertain at this time and requires carefully controlled studies and long-term follow-up.

  17. Occurrence of organohalogens at the Dead Sea Basin

    Science.gov (United States)

    Tubbesing, Christoph; Kotte, Karsten; Keppler, Frank; Krause, Torsten; Bahlmann, Enno; Schöler, Heinfried

    2013-04-01

    Most arid and semi-arid regions are characterized by evaporites, which are assured sources for volatile organohalogens (VOX) [1]. These compounds play an important role in tropospheric and stratospheric chemistry. The Dead Sea between Israel and Jordan is the world's most famous and biggest all-season water covered salt lake. In both countries chemical plants like the Dead Sea Works and the Arab Potash Company are located at the southern part of the Dead Sea and mine various elements such as bromine and magnesium. Conveying sea water through constructed evaporation pans multifarious salts are enriched and precipitated. In contrast, the Northern basin and main part of the Dead Sea has remained almost untouched by industrial salt production. Its fresh water supply from the Jordan River is constantly decreasing, leading to further increased salinity. During a HALOPROC campaign (Natural Halogenation Processes in the Environment) we collected various samples including air, soils, sediments, halophytic plants, ground- and seawater from the Northern and Southern basin of the Israeli side of the Dead Sea. These samples were investigated for the occurrence of halocarbons using different analytical techniques. Most samples were analyzed for volatile organohalogens such as haloalkanes using gas chromatography- mass spectrometry (GC-MS). Interestingly, there is a strong enrichment of trihalomethanes (THM), especially all chlorinated and brominated ones and also the iodinated compound dichloroiodomethane were found in the Southern basin. In addition, volatile organic carbons (VOC) such as ethene and some other alkenes were analyzed by a gas chromatography-flame ionisation detector (GC-FID) to obtain further information about potential precursors of halogenated compounds. Halophytic plants were investigated for their potential to release chloromethane and bromomethane but also for their stable carbon and hydrogen isotope composition. For this purpose, a plant chamber was

  18. The Enzyme Function Initiative†

    Science.gov (United States)

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  19. The Enzyme Function Initiative.

    Science.gov (United States)

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

    2011-11-22

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.

  20. DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

    Science.gov (United States)

    Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

    2014-12-20

    Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

  1. Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

    Directory of Open Access Journals (Sweden)

    Kim Sang-Hoon

    2012-02-01

    Full Text Available Abstract Background The genome of the Gram-positive, metal-reducing, dehalorespiring Desulfitobacterium hafniense DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of Desulfitobacterium hafniense Y51, the phylogenetically closest strain among the species with a sequenced genome. Results The genome of Desulfitobacterium hafniense DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N2 and CO2, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The D. hafniense DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO2-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress. Conclusions D. hafniense DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N2 and CO2 fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.

  2. Microbial reductive dehalogenation of trihalomethanes by a Dehalobacter-containing co-culture.

    Science.gov (United States)

    Zhao, Siyan; Rogers, Matthew J; He, Jianzhong

    2017-07-01

    Trihalomethanes such as chloroform and bromoform, although well-known as a prominent class of disinfection by-products, are ubiquitously distributed in the environment due to widespread industrial usage in the past decades. Chloroform and bromoform are particularly concerning, of high concentrations detected and with long half-lives up to several hundred days in soils and groundwater. In this study, we report a Dehalobacter- and Desulfovibrio-containing co-culture that exhibits dehalogenation of chloroform (~0.61 mM) to dichloromethane and bromoform (~0.67 mM) to dibromomethane within 10-15 days. This co-culture was further found to dechlorinate 1,1,1-trichloroethane (1,1,1-TCA) (~0.65 mM) to 1,1-dichloroethane within 12 days. The Dehalobacter species present in this co-culture, designated Dehalobacter sp. THM1, was found to couple growth with dehalogenation of chloroform, bromoform, and 1,1,1-TCA. Strain THM1 harbors a newly identified reductive dehalogenase (RDase), ThmA, which catalyzes chloroform, bromoform, and 1,1,1-TCA dehalogenation. Additionally, based on the sequences of thmA and other identified chloroform RDase genes, ctrA, cfrA, and tmrA, a pair of chloroform RDase gene-specific primers were designed and successfully applied to investigate the chloroform dechlorinating potential of microbial communities. The comparative analysis of chloroform RDases with tetrachloroethene RDases suggests a possible approach in predicting the substrate specificity of uncharacterized RDases in the future.

  3. Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

    Science.gov (United States)

    Ahn, Young-Beom; Rhee, Sung-Keun; Fennell, Donna E.; Kerkhof, Lee J.; Hentschel, Ute; Häggblom, Max M.

    2003-01-01

    Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration. PMID:12839794

  4. Yarrowia lipolytica NCIM 3589, a tropical marine yeast, degrades bromoalkanes by an initial hydrolytic dehalogenation step.

    Science.gov (United States)

    Vatsal, Aakanksha; Zinjarde, Smita S; Kumar, Ameeta Ravi

    2015-04-01

    The widespread industrial use of organobromines which are known persistent organic pollutants has led to their accumulation in sediments and water bodies causing harm to animals and humans. While degradation of organochlorines by bacteria is well documented, information regarding degradation pathways of these recalcitrant organobromines is scarce. Hence, their fates and effects on the environment are of concern. The present study shows that a tropical marine yeast, Yarrowia lipolytica NCIM 3589 aerobically degrades bromoalkanes differing in carbon chain length and position of halogen substitution viz., 2-bromopropane (2-BP), 1-bromobutane (1-BB), 1,5 dibromopentane (1,5-DBP) and 1-bromodecane (1-BD) as seen by an increase in cell mass, release of bromide and concomitant decrease in concentration of brominated compound. The amount of bromoalkane degraded was 27.3, 21.9, 18.0 and 38.3 % with degradation rates of 0.076, 0.058, 0.046 and 0.117/day for 2-BP, 1-BB, 1,5-DBP and 1-BD, respectively. The initial product formed respectively were alcohols viz., 2-propanol, 1-butanol, 1-bromo, 5-pentanol and 1-decanol as detected by GC-MS. These were further metabolized to fatty acids viz., 2-propionic, 1-butyric and 1-decanoic acid eventually leading to carbon dioxide formation. Neither higher chain nor brominated fatty acids were detected. An inducible extracellular dehalogenase responsible for removal of bromide was detected with activities of 21.07, 18.82, 18.96 and 26.67 U/ml for 2-BP, 1-BB, 1,5-DBP and 1-BD, respectively. We report here for the first time the proposed aerobic pathway of bromoalkane degradation by an eukaryotic microbe Y. lipolytica 3589, involving an initial hydrolytic dehalogenation step.

  5. Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Patrick K. H. [University of California, Berkeley; Dill, Brian [ORNL; Louie, Tiffany S. [University of California, Berkeley; Shah, Manesh B [ORNL; Verberkmoes, Nathan C [ORNL; Andersen, Gary L. [Lawrence Berkeley National Laboratory (LBNL); Zinder, Stephen H. [Cornell University; Alvarez-Cohen, Lisa [Lawrence Berkeley National Laboratory (LBNL)

    2012-01-01

    Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL) as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

  6. Microbial Community Response of an Organohalide Respiring Enrichment Culture to Permanganate Oxidation.

    Science.gov (United States)

    Sutton, Nora B; Atashgahi, Siavash; Saccenti, Edoardo; Grotenhuis, Tim; Smidt, Hauke; Rijnaarts, Huub H M

    2015-01-01

    While in situ chemical oxidation is often used to remediate tetrachloroethene (PCE) contaminated locations, very little is known about its influence on microbial composition and organohalide respiration (OHR) activity. Here, we investigate the impact of oxidation with permanganate on OHR rates, the abundance of organohalide respiring bacteria (OHRB) and reductive dehalogenase (rdh) genes using quantitative PCR, and microbial community composition through sequencing of 16S rRNA genes. A PCE degrading enrichment was repeatedly treated with low (25 μmol), medium (50 μmol), or high (100 μmol) permanganate doses, or no oxidant treatment (biotic control). Low and medium treatments led to higher OHR rates and enrichment of several OHRB and rdh genes, as compared to the biotic control. Improved degradation rates can be attributed to enrichment of (1) OHRB able to also utilize Mn oxides as a terminal electron acceptor and (2) non-dechlorinating community members of the Clostridiales and Deltaproteobacteria possibly supporting OHRB by providing essential co-factors. In contrast, high permanganate treatment disrupted dechlorination beyond cis-dichloroethene and caused at least a 2-4 orders of magnitude reduction in the abundance of all measured OHRB and rdh genes, as compared to the biotic control. High permanganate treatments resulted in a notably divergent microbial community, with increased abundances of organisms affiliated with Campylobacterales and Oceanospirillales capable of dissimilatory Mn reduction, and decreased abundance of presumed supporters of OHRB. Although OTUs classified within the OHR-supportive order Clostridiales and OHRB increased in abundance over the course of 213 days following the final 100 μmol permanganate treatment, only limited regeneration of PCE dechlorination was observed in one of three microcosms, suggesting strong chemical oxidation treatments can irreversibly disrupt OHR. Overall, this detailed investigation into dose

  7. Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

    Science.gov (United States)

    Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

    2015-12-04

    In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A global view of structure-function relationships in the tautomerase superfamily.

    Science.gov (United States)

    Davidson, Rebecca; Baas, Bert-Jan; Akiva, Eyal; Holliday, Gemma L; Polacco, Benjamin J; LeVieux, Jake A; Pullara, Collin R; Zhang, Yan Jessie; Whitman, Christian P; Babbitt, Patricia C

    2018-02-16

    The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis -3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase-like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Transports of acetate and haloacetate in Burkholderia species MBA4 are operated by distinct systems

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    Su Xianbin

    2012-11-01

    Full Text Available Abstract Background Acetate is a commonly used substrate for biosynthesis while monochloroacetate is a structurally similar compound but toxic and inhibits cell metabolism by blocking the citric acid cycle. In Burkholderia species MBA4 haloacetate was utilized as a carbon and energy source for growth. The degradation of haloacid was mediated by the production of an inducible dehalogenase. Recent studies have identified the presence of a concomitantly induced haloacetate-uptake activity in MBA4. This uptake activity has also been found to transport acetate. Since acetate transporters are commonly found in bacteria it is likely that haloacetate was transported by such a system in MBA4. Results The haloacetate-uptake activity of MBA4 was found to be induced by monochloroacetate (MCA and monobromoacetate (MBA. While the acetate-uptake activity was also induced by MCA and MBA, other alkanoates: acetate, propionate and 2-monochloropropionate (2MCPA were also inducers. Competing solute analysis showed that acetate and propionate interrupted the acetate- and MCA- induced acetate-uptake activities. While MCA, MBA, 2MCPA, and butyrate have no effect on acetate uptake they could significantly quenched the MCA-induced MCA-uptake activity. Transmembrane electrochemical potential was shown to be a driving force for both acetate- and MCA- transport systems. Conclusions Here we showed that acetate- and MCA- uptake in Burkholderia species MBA4 are two transport systems that have different induction patterns and substrate specificities. It is envisaged that the shapes and the three dimensional structures of the solutes determine their recognition or exclusion by the two transport systems.

  10. Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily

    International Nuclear Information System (INIS)

    Burks, Elizabeth A.; Fleming, Christopher D.; Mesecar, Andrew D.; Whitman, Christian P.; Pegan, Scott D.

    2010-01-01

    4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three αβ dimers. Like the P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the β-α-β building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.

  11. Interest of nuclear medicine in the diagnosis of congenital and childhood hypothyroidism; Apports de la medecine nucleaire au diagnostic des hypothyroidies congenitales et de l'enfant

    Energy Technology Data Exchange (ETDEWEB)

    Clerc, J. [Hopital Necker, Medecine Nucleaire, 75 - Paris (France)

    2002-08-01

    The paediatric thyroid scan (PTS) remains a cornerstone test in guiding the etiological diagnosis of congenital hypothyroidism (CH). In hypothyroid babies, thyroxine therapy must be started immediately. A reliable PTS can be obtained in the following days even under T4 therapy. {sup 123}I is the isotope of choice since it provides quantitated images and a clinically relevant grading of dys-hormonal-genetic disorders. The dosimetry of {sup 123}I is lower than usually considered because iodine uptake is absent or low in most cases of CH and because the energy deposited within the colloid has no expected radiobiological detriment. PTS is a highly contributive, sensitive and reproducible test in identifying thyroid dysgenesis the most frequent etiology (70%) of permanent CH. Since agenesis requires a very careful T4 therapy monitoring, PTS is also of therapeutic interest. PTS can distinguish 3 types of dys-hormonal-genetic disorders (10%). In type 1, low uptake indicates a defective R-TSH or a NIS defect when {sup 123}I gastric uptake is absent. Type 2 - high uptake, goiter and positive perchlorate discharge test (PDT) - refers to organification defects (TPO, THOX1,2, Pendrin). Type 3 (goiter, high uptake, negative PDT) includes coupling defects, thyroglobulin abnormalities and dehalogenase deficiency. Main transient aetiologies (20%) of CH are iodine overload and blocking anti hR-TSH antibodies, while thyroiditis and dietary iodine overload are more frequent thereafter The molecular understanding of the defects involved in CH rapidly develops. However, precocious diagnosis, appropriate T4 therapy and sorting out the etiology are the most relevant parameters which determine the final clinical prognosis. (author)

  12. Marine microbe with potential to adhere and degrade plastic structures

    Directory of Open Access Journals (Sweden)

    Alka Kumari

    2017-10-01

    Full Text Available Extensive usages of plastics have led to their accumulation as a contaminant in natural environment worldwide. Plastic is an inert and non-biodegradable material, due to its complex structure and hydrophobic backbone [1]. Conventional methods for reduction of plastic waste such as burning, land-filling release unwanted toxic chemicals to the environment and harming living organism of land as well as the ocean. There is growing interest in development of strategies for the degradation of plastic wastes to clean the environment [2]. Marine bacteria have evolved with the capability to adapt and grow in the diverse environmental conditions [3]. We studied the ability of marine bacteria for destabilization and utilization of different plastic films (LDPE, HDPE, PVC and PET as a sole source of carbon. An active bacterial strain AIIW2 was selected based on the triphenyl tetrazolium chloride reduction assay, and it was identified as Bacillus species based on 16S rRNA gene sequence. The viability of the strain over the plastic surface was studied and confirmed by bacLight assay with fluorescent probes. Scanning Electron Microscope and Atomic Force Microscope images suggested that bacterial interaction over the plastic surface is causing deterioration and roughness with increasing bacterial incubation time. In Fourier transform infrared spectra of treated plastic film evidenced stretching of the (-CH alkane rock chain and (-CO carbonyl region, suggested the oxidative activities of the bacteria. The results revealed that ability of bacterial strain for instigating their colonization over plastic films and deteriorating the polymeric structure in the absence of other carbon sources [4]. Moreover, production of extracellular enzymes such as esterase, laccase, and dehalogenase which are reported to support utilization of plastics was confirmed by plate assays. In concise, our results suggested that the marine bacterial strain AIIW2 have the ability to utilize

  13. Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes

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    Holly L. Sewell

    2017-12-01

    Full Text Available The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4 in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2 as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA in the phylum Chloroflexi (DscP3 and Dsc4, including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187 of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of

  14. Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes.

    Science.gov (United States)

    Sewell, Holly L; Kaster, Anne-Kristin; Spormann, Alfred M

    2017-12-19

    The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive

  15. Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium

    Directory of Open Access Journals (Sweden)

    Pauline Chaignaud

    2017-09-01

    Full Text Available Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially

  16. Synthesis, solubilization, and surface functionalization of highly fluorescent quantum dots for cellular targeting through a small molecule

    Science.gov (United States)

    Galloway, Justin F.

    modified dehalogenase, was added to the N-terminus of the cAR1 receptor without resulting in a phenotype. The Halo protein fused to cAR1 was then shown to bind an organic fluorophore by the cleavage of a chloroalkane bond. Though QDs functionalized with a chloroalkane were able to bind free Halo protein, no specific binding to the Halo protein fused to cAR1 was observed.

  17. Chlorinated Electron Acceptor Abundance Drives Selection of Dehalococcoides mccartyi (D. mccartyi Strains in Dechlorinating Enrichment Cultures and Groundwater Environments

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    Alfredo Pérez-de-Mora

    2018-05-01

    Full Text Available Dehalococcoides mccartyi (D. mccartyi strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA genes within their respective genomes. While multiple rdhA genes have been sequenced, the activity of the corresponding proteins has been identified in only a few cases. Examples include the enzymes whose substrates are groundwater contaminants such as trichloroethene (TCE, cis-dichloroethene (cDCE and vinyl chloride (VC. The associated rdhA genes, namely tceA, bvcA, and vcrA, along with the D. mccartyi 16S rRNA gene are often used as biomarkers of growth in field samples. In this study, we monitored an additional 12 uncharacterized rdhA sequences identified in the metagenome in the mixed D. mccartyi-containing culture KB-1 to monitor population shifts in more detail. Quantitative PCR (qPCR assays were developed for 15 D. mccartyi rdhA genes and used to measure population diversity in 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion of rdhA gene copies relative to D. mccartyi 16S rRNA gene copies revealed the presence of multiple distinct D. mccartyi strains in each culture, many more than the two strains inferred from 16S rRNA analysis. The specific electron acceptor amended to each culture had a major influence on the distribution of D. mccartyi strains and their associated rdhA genes. We also surveyed the abundance of rdhA genes in samples from two bioaugmented field sites (Canada and United Kingdom. Growth of the dominant D. mccartyi strain in KB-1 was detected at the United Kingdom site. At both field sites, the measurement of relative rdhA abundances revealed D. mccartyi population shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene. These shifts indicate a selective pressure of the most abundant chlorinated electron acceptor, as was also observed in lab cultures. These

  18. Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

    Directory of Open Access Journals (Sweden)

    Gretchen L. W. Heavner

    2018-02-01

    Full Text Available Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10−12 to 5.9 × 10−10 microelectron equivalents per cell per hour (μeeq/cell∙h. Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA and the hydrogenase HupL (R2 = 0.83 and 0.88, respectively, but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration

  19. Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE-DNAPL source zone.

    Science.gov (United States)

    Cápiro, Natalie L; Löffler, Frank E; Pennell, Kurt D

    2015-11-01

    Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE-DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis-1,2-dichloroethene (cis-DCE) formation after 1 PV (ca. 7 days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90 μM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0±1.3 and 4.0±1.7 fold in the pool and ganglia regions, respectively. Dhc and GeoSZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE-DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥155 μM) and ethene (≥65 μM) concentrations were measured in zones where Dhc and GeoSZ were predominately attached to the solids. These findings demonstrate

  20. Optimization of synthesis and quality control procedures for the preparations of 18F and 123I labelled peptides

    International Nuclear Information System (INIS)

    Archimandritis, S.C.; Potamianos, S.; Varvarigou, A.D.

    2002-01-01

    Radiolabelled biomolecules like proteins and peptides, are playing now days an important role in experimental and clinical Nuclear Medicine. Radioiodination techniques remain important, with improvements accounting for high purity, specific activity and better in vivo stability. Radioiodination using prosthetic groups is the method of choice in cases where the molecules are lacking of thyrosyl groups in their structure and are also sensitive to circulating dehalogenase enzymes. This investigation was based on the need to optimize labelling and quality control techniques for these molecules. The N-succinimidyliodobenzoate (SIB) was used in this study as the prosthetic group for the radioiodination. Optimization of SIB synthesis and modification of the protocol resulted in an improved mean yield of SIB. The combination of TLC and column chromatography using silica gel proved suitable in identifying SIB. Furthermore, the ability of SIB to couple to protein was also used to confirm the presence of SIB. In this case, SEC and ITLC-SG proved suitable to confirm protein binding of SIB. Column chromatography using silica gel containing Sep-Pak was appropriate for SIB purification. Concerning SIB conjugation to peptides, high radioiodination yields were only possible for peptides with amino-containing-side-chain amino acids. Furthermore, lysine containing peptides retained stability, at 4 deg. C, for at least 24 h and reverse phase HPLC proved the most suitable technique for assessing conjugation of SIB to peptide. The biological evaluation of the radiolabelled product was made in normal mice. SIB and SIB-peptide conjugates were tested comparatively and a number of tentative but interesting inferences were drawn. SIB and its peptide conjugates exhibited good in vivo stability as evidenced by low thyroid accumulation and were cleared via the kidneys. A time dependant decrease in the% dose per gram of tissue indicates possible adrenal metabolism of SIB and SIB

  1. Biological Chlorine Cycling in Arctic Peat Soils

    Science.gov (United States)

    Zlamal, J. E.; Raab, T. K.; Lipson, D.

    2014-12-01

    Soils of the Arctic tundra near Barrow, Alaska are waterlogged and anoxic throughout most of the profile due to underlying permafrost. Microbial communities in these soils are adapted for the dominant anaerobic conditions and are capable of a surprising diversity of metabolic pathways. Anaerobic respiration in this environment warrants further study, particularly in the realm of electron cycling involving chlorine, which preliminary data suggest may play an important role in arctic anaerobic soil respiration. For decades, Cl was rarely studied outside of the context of solvent-contaminated sites due to the widely held belief that it is an inert element. However, Cl has increasingly become recognized as a metabolic player in microbial communities and soil cycling processes. Organic chlorinated compounds (Clorg) can be made by various organisms and used metabolically by others, such as serving as electron acceptors for microbes performing organohalide respiration. Sequencing our arctic soil samples has uncovered multiple genera of microorganisms capable of participating in many Cl-cycling processes including organohalide respiration, chlorinated hydrocarbon degradation, and perchlorate reduction. Metagenomic analysis of these soils has revealed genes for key enzymes of Cl-related metabolic processes such as dehalogenases and haloperoxidases, and close matches to genomes of known organohalide respiring microorganisms from the Dehalococcoides, Dechloromonas, Carboxydothermus, and Anaeromyxobacter genera. A TOX-100 Chlorine Analyzer was used to quantify total Cl in arctic soils, and these data were examined further to separate levels of inorganic Cl compounds and Clorg. Levels of Clorg increased with soil organic matter content, although total Cl levels lack this trend. X-ray Absorption Near Edge Structure (XANES) was used to provide information on the structure of Clorg in arctic soils, showing great diversity with Cl bound to both aromatic and alkyl groups

  2. Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

    Science.gov (United States)

    Heavner, Gretchen L W; Mansfeldt, Cresten B; Debs, Garrett E; Hellerstedt, Sage T; Rowe, Annette R; Richardson, Ruth E

    2018-02-08

    Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1 TM , including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1 TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1 TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10 -12 to 5.9 × 10 -10 microelectron equivalents per cell per hour (μeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1 TM . Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1 TM . The addition of oxygen induced cell stress that caused respiration rates