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Jiri Damborsky - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and Crystallographic Analysis of a Bradyrhizobium Elkanii USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site
Crystals, 2019Co-Authors: Tatyana Prudnikova, Jiri Damborsky, Radka Chaloupkova, Barbora Kascakova, Jeroen R. Mesters, P. Grinkevich, P. Havlickova, Andrii Mazur, Anastasiia Shaposhnikova, M. KutyAbstract:Haloalkane Dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the Haloalkane Dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeAΔCl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeAΔCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeAΔCl has been determined and refined to the 1.4 A resolution. The DbeAΔCl crystals belong to monoclinic space group C121. The DbeAΔCl molecular structure was characterized and compared with five known Haloalkane Dehalogenases selected from the Protein Data Bank.
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Crystal structure of the cold-adapted Haloalkane Dehalogenase DpcA from Psychrobacter cryohalolentis K5.
Acta crystallographica. Section F Structural biology communications, 2019Co-Authors: Katsiaryna Tratsiak, Ivana Drienovska, Jiri Damborsky, Radka Chaloupkova, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Ivana Kuta SmatanovaAbstract:Haloalkane Dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, Haloalkane Dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the Haloalkane Dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
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Crystal structure of the cold‐adapted Haloalkane Dehalogenase DpcA from Psychrobacter cryohalolentis K5
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2019Co-Authors: Katsiaryna Tratsiak, Ivana Drienovska, Jiri Damborsky, Radka Chaloupkova, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Ivana Kuta SmatanovaAbstract:Haloalkane Dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, Haloalkane Dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the Haloalkane Dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure–function relationships of cold-adapted enzymes.
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conformational changes allow processing of bulky substrates by a Haloalkane Dehalogenase with a small and buried active site
Journal of Biological Chemistry, 2018Co-Authors: Piia Kokkonen, Zbynek Prokop, David Bednar, Veronika Dockalova, Jiri DamborskyAbstract: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 angstrom(3)) 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 angstrom(3)). 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 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 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.
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a Haloalkane Dehalogenase from a marine microbial consortium possessing exceptionally broad substrate specificity
Applied and Environmental Microbiology, 2017Co-Authors: Tomas Buryska, Jiri Damborsky, Petra Babkova, Ondrej Vavra, Zbynek ProkopAbstract:ABSTRACT The Haloalkane Dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized Haloalkane Dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the Haloalkane Dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.
Dick B Janssen - One of the best experts on this subject based on the ideXlab platform.
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The Haloalkane Dehalogenase Genes DHLA and dhaA are Globally Distributed and Highly Conserved
Biotechnology for the Environment: Strategy and Fundamentals, 2020Co-Authors: Gerrit J Poelarends, Tjibbe Bosma, Johan E. T. Van Hylckama Vlieg, Dick B JanssenAbstract:Direct hydrolytic dehalogenation by Haloalkane Dehalogenases is the most important mechanism involved in biodegradation of synthetic Haloalkanes that occur as soil pollutants. Here we show that five Gram-negative 1,2-dichloroethane-utilizing bacteria contain Haloalkane Dehalogenase genes identical to the dhlA gene from Xanthobacter autotrophicus GJ10, whereas five Gram-positive chloroalkane degraders, independently isolated from geographically distinct locations, contain genes identical to the dhaA gene of Rhodococcus rhodochrous NCIMB13064. Furthermore, the dhaA gene was detected by PCR amplification in fifteen newly isolated Gram-positive chloroalkane-degrading bacteria. Our results suggest that the dhlA and dhaA genes recently arose from a single origin and have become distributed globally, most likely as the result of the massive and worldwide use of synthetic chlorinated hydrocarbons in industry and agriculture.
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regio and enantioselective sequential dehalogenation of rac 1 3 dibromobutane by Haloalkane Dehalogenase linb
ChemBioChem, 2016Co-Authors: Johannes Gross, Zbyněk Prokop, Dick B Janssen, Kurt Faber, Melanie HallAbstract:The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by Haloalkane Dehalogenase LinB fromSphingobium japonicum UT26 proceeds in a sequential fashion via 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. Due to complex competition between all eight possible pathways, intermediate haloalcohols could be formed with moderate to good ee values [(S)-4-bromobutan-2-ol in up to 87% ee]. Similarly, (S)-1,3-butanediol was formed in max. ee 35% before full hydrolysis furnished the racemic diol product.
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computational library design for increasing Haloalkane Dehalogenase stability
ChemBioChem, 2014Co-Authors: Robert J Floor, Wiktor Szymanski, Ben L Feringa, Hein J Wijma, Dana I Colpa, Aline Ramossilva, Peter A Jekel, Siewert J Marrink, Dick B JanssenAbstract:We explored the use of a computational design framework for the stabilization of the Haloalkane Dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 degrees C increase in apparent melting temperature (T-m,T-app, 72.5 degrees C) and an over 200-fold longer half-life at 60 degrees C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.
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dynamic kinetic resolution process employing Haloalkane Dehalogenase
ACS Catalysis, 2011Co-Authors: Alja Westerbeek, Wiktor Szymanski, Ben L Feringa, Dick B JanssenAbstract:The first dynamic kinetic resolution process with a Haloalkane Dehalogenase is described, allowing the efficient preparation of enantiopure α-hydroxyamides from racemic α-bromoamides. A simple membrane reactor is used to separate the enzyme from the nonsoluble, polymer-based, and metal-free racemizing agent. A model substrate, N-phenyl-2-bromopropionamide, was converted to (S)-N-phenyl-2-hydroxypropionamide either with 63% yield and 95% e.e or with 78% yield and 88% e.e.
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thermodynamic analysis of halide binding to Haloalkane Dehalogenase suggests the occurrence of large conformational changes
Protein Science, 2008Co-Authors: Geja H Krooshof, Armand W J W Tepper, Rene Floris, Dick B JanssenAbstract:Haloalkane Dehalogenase (DhlA) hydrolyzes short-chain Haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three-step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three-step route involves different and perhaps larger conformational changes than the two-step export route. We propose that the three-step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two-step route for halide release involves the transfer of the halide ion from the halide-binding site in the cavity to a binding site somewhere at the protein surface, where a so-called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements are necessary for this latter process.
Yuji Nagata - One of the best experts on this subject based on the ideXlab platform.
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Protein engineering of Haloalkane Dehalogenase LinB:reconstruction of active site and modification of entrancetunnel
2020Co-Authors: Marta Monincova, Radka Chaloupkova, Yuji Nagata, Zbyněk Prokop, Masataka Tsuda, Andrea Fořtová, Martina Pavlová, Jiři DamborskýAbstract:Haloalkane Dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited Dehalogenase activity with the Haloalkanes tested, confirming that Rv2579 is a member of the Haloalkane Dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of Haloalkane Dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in Haloalkane Dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.
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Determination of Haloalkane Dehalogenase enzymatic activity bycapillary zone electrophoresis
Journal of Chromatography A, 2020Co-Authors: Zdeněk Glatz, Jiři Damborský, Michaela Wimmerová, María Victoria Marini Palomeque, Yuji NagataAbstract:A new sensitive method has been developed for the determination of Haloalkane Dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monito red by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 mm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity ) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M b-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in bot cases 25oC. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.
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Determination of Haloalkane Dehalogenase activity by capillary zone
2020Co-Authors: Zdenek Glatz, Maria V. Marini, Michaela Wimmerová, Yuji NagataAbstract:A new sensitive method has been developed for the determination of Haloalkane Dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product — bromide or chloride ions — was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 mm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M b-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 258C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate. © 2000 Elsevier Science B.V. All rights reserved.
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the effect of a unique halide stabilizing residue on the catalytic properties of Haloalkane Dehalogenase data from agrobacterium tumefaciens c58
FEBS Journal, 2013Co-Authors: Khomaini Hasan, Jiri Damborsky, Radka Chaloupkova, Yuji Nagata, Andrea Fortova, Jan Brezovsky, Artur Gora, Hana Moskalikova, Zbynek ProkopAbstract:Haloalkane Dehalogenases catalyze the hydrolysis of carbon–halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known Haloalkane Dehalogenases, these residues are either a pair of tryptophans or a tryptophan–asparagine pair. The newly-isolated Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K0.5 for selected halogenated substrates by a factor of 2–4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural Haloalkane Dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.
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crystallization and preliminary x ray analysis of the Haloalkane Dehalogenase data from agrobacterium tumefaciens c58
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: Tomoko Mase, Yuji Nagata, Hideya Yabuki, Masahiko Okai, Jun Ohtsuka, Fabiana Lica Imai, Masaru TanokuraAbstract: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 A 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 A. The crystal contained two molecules in the asymmetric unit.
Jiři Damborský - One of the best experts on this subject based on the ideXlab platform.
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Determination of Haloalkane Dehalogenase enzymatic activity bycapillary zone electrophoresis
Journal of Chromatography A, 2020Co-Authors: Zdeněk Glatz, Jiři Damborský, Michaela Wimmerová, María Victoria Marini Palomeque, Yuji NagataAbstract:A new sensitive method has been developed for the determination of Haloalkane Dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monito red by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 mm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity ) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M b-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in bot cases 25oC. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.
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Optical biosensor based on Haloalkane Dehalogenase fordetection of halogenated hydrocarbons in the environment.
2020Co-Authors: Šárka Bidmanová, Radka Chaloupkova, Zbyněk Prokop, Jiři DamborskýAbstract:Poster " Optical biosensor based on Haloalkane Dehalogenase for detection of halogenated hydrocarbons in the environment" presented by Mgr. Sarka Bidmanova on conference "Biosensors" in Glasgow.
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Protein engineering of Haloalkane Dehalogenase LinB:reconstruction of active site and modification of entrancetunnel
2020Co-Authors: Marta Monincova, Radka Chaloupkova, Yuji Nagata, Zbyněk Prokop, Masataka Tsuda, Andrea Fořtová, Martina Pavlová, Jiři DamborskýAbstract:Haloalkane Dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited Dehalogenase activity with the Haloalkanes tested, confirming that Rv2579 is a member of the Haloalkane Dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of Haloalkane Dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in Haloalkane Dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.
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immobilization of Haloalkane Dehalogenase linb from sphingobium japonicum ut26 for biotechnological applications
Journal of Biocatalysis & Biotransformation, 2013Co-Authors: Šárka Bidmanová, Jiři Damborský, Zbyněk ProkopAbstract:Immobilization of Haloalkane Dehalogenase LinB from Sphingobium japonicum UT26 for Biotechnological Applications Haloalkane Dehalogenases are enzymes capable of converting a broad range of aliphatic halogenated compounds to corresponding alcohols. These Dehalogenase-based biotransformations are attractive for various biological processes, e.g. biocatalysis, bioremediation and detoxification, which often require protein immobilization.
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Degradation of β-hexachlorocyclohexane by Haloalkane Dehalogenase LinB from γ-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205
Archives of Microbiology, 2007Co-Authors: Zbynek Prokop, Masataka Tsuda, Jiři Damborský, Martin Klvaňa, Yoshiyuki Otsubo, Yuji NagataAbstract:The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal γ-isomer and noninsecticidal α-, β-, and δ-isomers, among which β-HCH is the most recalcitrant and has caused serious environmental problems. A γ-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded β-HCH more rapidly than the well-characterized γ-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, β-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A Haloalkane Dehalogenase LinB (LinB_MI) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB_UT) was identified as an enzyme responsible for the two-step conversion of β-HCH to TCDL. This property of LinB_MI contrasted with that of LinB_UT, which catalyzed only the first step conversion of β-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB_MI. However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB_MI. Sequence analysis revealed that MI1205 possesses the IS 6100 -flanked cluster that contains two copies of the linB _MI gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.
Radka Chaloupkova - One of the best experts on this subject based on the ideXlab platform.
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Optical biosensor based on Haloalkane Dehalogenase fordetection of halogenated hydrocarbons in the environment.
2020Co-Authors: Šárka Bidmanová, Radka Chaloupkova, Zbyněk Prokop, Jiři DamborskýAbstract:Poster " Optical biosensor based on Haloalkane Dehalogenase for detection of halogenated hydrocarbons in the environment" presented by Mgr. Sarka Bidmanova on conference "Biosensors" in Glasgow.
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Protein engineering of Haloalkane Dehalogenase LinB:reconstruction of active site and modification of entrancetunnel
2020Co-Authors: Marta Monincova, Radka Chaloupkova, Yuji Nagata, Zbyněk Prokop, Masataka Tsuda, Andrea Fořtová, Martina Pavlová, Jiři DamborskýAbstract:Haloalkane Dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited Dehalogenase activity with the Haloalkanes tested, confirming that Rv2579 is a member of the Haloalkane Dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of Haloalkane Dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in Haloalkane Dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.
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Crystallization and Crystallographic Analysis of a Bradyrhizobium Elkanii USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site
Crystals, 2019Co-Authors: Tatyana Prudnikova, Jiri Damborsky, Radka Chaloupkova, Barbora Kascakova, Jeroen R. Mesters, P. Grinkevich, P. Havlickova, Andrii Mazur, Anastasiia Shaposhnikova, M. KutyAbstract:Haloalkane Dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the Haloalkane Dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeAΔCl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeAΔCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeAΔCl has been determined and refined to the 1.4 A resolution. The DbeAΔCl crystals belong to monoclinic space group C121. The DbeAΔCl molecular structure was characterized and compared with five known Haloalkane Dehalogenases selected from the Protein Data Bank.
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Crystal structure of the cold-adapted Haloalkane Dehalogenase DpcA from Psychrobacter cryohalolentis K5.
Acta crystallographica. Section F Structural biology communications, 2019Co-Authors: Katsiaryna Tratsiak, Ivana Drienovska, Jiri Damborsky, Radka Chaloupkova, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Ivana Kuta SmatanovaAbstract:Haloalkane Dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, Haloalkane Dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the Haloalkane Dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
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Crystal structure of the cold‐adapted Haloalkane Dehalogenase DpcA from Psychrobacter cryohalolentis K5
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2019Co-Authors: Katsiaryna Tratsiak, Ivana Drienovska, Jiri Damborsky, Radka Chaloupkova, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Ivana Kuta SmatanovaAbstract:Haloalkane Dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, Haloalkane Dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the Haloalkane Dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure–function relationships of cold-adapted enzymes.
