The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Jiri Damborsky - One of the best experts on this subject based on the ideXlab platform.
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Comparison of catalysis by haloalkane dehalogenases in aqueous solutions of deep eutectic and organic solvents
Green Chemistry, 2020Co-Authors: Veronika Stepankova, Jiri Damborsky, Pavel Vanacek, Radka ChaloupkováAbstract:Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds in diverse halogenated hydrocarbons and are attractive catalysts for sustainable biotechnologies. However, their use in industrial processes is limited due to the poor water solubility of their substrates and the tendency of the substrates to undergo abiotic hydrolysis. Here we systematically and critically compare the performance of three haloalkane dehalogenases, DbjA, DhaA and LinB, in aqueous solutions of the deep eutectic solvent ethaline, its components (ethylene glycol and choline chloride), and two organic solvents (methanol and acetone). Each of the solvents had different effects on the activity of each enzyme. Haloalkane dehalogenase DhaA was found to be the most tolerant to ethaline, retaining 21% of its reference activity even in solutions containing 90% (v/v) of ethaline. However, dissolution in 75% (v/v) ethylene glycol, 50% (v/v) methanol, or 25% (v/v) acetone caused almost total loss of DhaA activity. In contrast, the activities of DbjA and LinB were higher in ethylene glycol than in ethaline, and moreover the activity of DbjA was 1.5 times higher in 50% (v/v) ethylene glycol than in pure buffer. Interestingly, the enantioselectivity of 2-bromopentane hydrolysis catalysed by DbjA increased more than 4-fold in the presence of ethaline or ethylene glycol. Our results demonstrate that ethylene glycol and an ethylene glycol-based deep eutectic solvent can have beneficial effects on catalysis by haloalkane dehalogenases, broadening their usability in “green” biotechnologies.
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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, Jeroen R. Mesters, Barbora Kascakova, P. Grinkevich, P. Havlickova, Andrii Mazur, Anastasiia Shaposhnikova, Jiri Damborsky, Radka Chaloupková, Michal KutýAbstract: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 and Crystallization Communications, 2019Co-Authors: Katsiaryna Tratsiak, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Jiri Damborsky, Ivana Drienovska, Radka Chaloupková, 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 communications, 2019Co-Authors: Katsiaryna Tratsiak, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Jiri Damborsky, Ivana Drienovska, Radka Chaloupková, 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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a haloalkane dehalogenase from a marine microbial consortium possessing exceptionally broad substrate specificity
Applied and Environmental Microbiology, 2017Co-Authors: Tomas Buryska, Petra Babkova, Ondrej Vavra, Jiri Damborsky, 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.
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, Zbyněk Prokop, Andrea Fořtová, Martina Pavlová, Yuji Nagata, Masataka Tsuda, Radka Chaloupková, Jiří 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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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, Andrea Fortova, Artur Gora, Hana Moskalikova, Jan Brezovský, Jiri Damborsky, Yuji Nagata, Radka Chaloupková, 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, Hideya Yabuki, Jun Ohtsuka, Fabiana Lica Imai, Yuji Nagata, Masahiko Okai, 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.
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biochemical characteristics of the novel haloalkane dehalogenase data isolated from the plant pathogen agrobacterium tumefaciens c58
Applied and Environmental Microbiology, 2011Co-Authors: Khomaini Hasan, Andrea Fortova, Mayuko Ishitsuka, Tana Koudelakova, Jiri Damborsky, Yuji Nagata, Radka Chaloupková, Zbynek ProkopAbstract:We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.
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crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase dbja from bradyrhizobium japonicum usda110
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007Co-Authors: Yukari Sato, Ryo Natsume, Jiri Damborsky, Yuji Nagata, Masataka Tsuda, Toshiya SendaAbstract:Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. The haloalkane dehalogenase DbjA constitutes a novel substrate-specificity class with high catalytic activity for β-methylated haloalkanes. In order to reveal the mechanism of its substrate specificity, DbjA has been crystallized using the hanging-drop vapour-diffusion method. The best crystals were obtained using the microseeding technique with a reservoir solution consisting of 17–19.5%(w/v) PEG 4000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 7.7–8.0. The space group of the DbjA crystal is P21212, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 A. The crystal diffracts to 1.75 A resolution.
Jiří Damborský - 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, Zbyněk Prokop, Andrea Fořtová, Martina Pavlová, Yuji Nagata, Masataka Tsuda, Radka Chaloupková, Jiří 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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Optical biosensor based on haloalkane dehalogenase fordetection of halogenated hydrocarbons in the environment.
2020Co-Authors: Sarka Bidmanova, Zbyněk Prokop, Radka Chaloupková, Jiří 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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Haloalkane Dehalogenases From Marine Organisms
Methods in enzymology, 2018Co-Authors: Antonin Kunka, Jiří Damborský, Zbyněk ProkopAbstract:Haloalkane dehalogenases degrade halogenated compounds to corresponding alcohols by a hydrolytic mechanism. These enzymes are being intensively investigated as model systems in experimental and in silico studies of enzyme mechanism and evolution, but also hold importance as useful biocatalysts for a number of biotechnological applications. Haloalkane dehalogenases originate from various organisms including bacteria (degraders, symbionts, or pathogens), eukaryotes, and archaea. Several members of this enzyme family have been found in marine organisms. The marine environment represents a good source of enzymes with novel properties, because of its diverse living conditions. A number of novel dehalogenases isolated from marine environments show interesting characteristics such as high activity, unusually broad substrate specificity, stability, or selectivity. In this chapter, the overview of haloalkane dehalogenases from marine organisms is presented and their characteristics are summarized together with an overview of the methods for their identification and biochemical characterization.
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immobilization of haloalkane dehalogenase linb from sphingobium japonicum ut26 for biotechnological applications
Journal of Biocatalysis & Biotransformation, 2013Co-Authors: Sarka Bidmanova, Jiří 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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Nanosecond Time-Dependent Stokes Shift at the Tunnel Mouth of Haloalkane Dehalogenases
Journal of the American Chemical Society, 2009Co-Authors: Andrea Jesenska, Jan Brezovský, Jan Sýkora, Agnieszka Olżyńska, Zbyněk Zdráhal, Jiří DamborskýAbstract:The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide−hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obta...
Radka Chaloupková - 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: Sarka Bidmanova, Zbyněk Prokop, Radka Chaloupková, Jiří 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, Zbyněk Prokop, Andrea Fořtová, Martina Pavlová, Yuji Nagata, Masataka Tsuda, Radka Chaloupková, Jiří 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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Comparison of catalysis by haloalkane dehalogenases in aqueous solutions of deep eutectic and organic solvents
Green Chemistry, 2020Co-Authors: Veronika Stepankova, Jiri Damborsky, Pavel Vanacek, Radka ChaloupkováAbstract:Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds in diverse halogenated hydrocarbons and are attractive catalysts for sustainable biotechnologies. However, their use in industrial processes is limited due to the poor water solubility of their substrates and the tendency of the substrates to undergo abiotic hydrolysis. Here we systematically and critically compare the performance of three haloalkane dehalogenases, DbjA, DhaA and LinB, in aqueous solutions of the deep eutectic solvent ethaline, its components (ethylene glycol and choline chloride), and two organic solvents (methanol and acetone). Each of the solvents had different effects on the activity of each enzyme. Haloalkane dehalogenase DhaA was found to be the most tolerant to ethaline, retaining 21% of its reference activity even in solutions containing 90% (v/v) of ethaline. However, dissolution in 75% (v/v) ethylene glycol, 50% (v/v) methanol, or 25% (v/v) acetone caused almost total loss of DhaA activity. In contrast, the activities of DbjA and LinB were higher in ethylene glycol than in ethaline, and moreover the activity of DbjA was 1.5 times higher in 50% (v/v) ethylene glycol than in pure buffer. Interestingly, the enantioselectivity of 2-bromopentane hydrolysis catalysed by DbjA increased more than 4-fold in the presence of ethaline or ethylene glycol. Our results demonstrate that ethylene glycol and an ethylene glycol-based deep eutectic solvent can have beneficial effects on catalysis by haloalkane dehalogenases, broadening their usability in “green” biotechnologies.
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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, Jeroen R. Mesters, Barbora Kascakova, P. Grinkevich, P. Havlickova, Andrii Mazur, Anastasiia Shaposhnikova, Jiri Damborsky, Radka Chaloupková, Michal KutýAbstract: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 and Crystallization Communications, 2019Co-Authors: Katsiaryna Tratsiak, Michal Kutý, Tatyana Prudnikova, Jiri Brynda, Petr Pachl, Pavlina Rezacova, Jiri Damborsky, Ivana Drienovska, Radka Chaloupková, 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.
M. W. Anders - One of the best experts on this subject based on the ideXlab platform.
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Formation and Fate of Reactive Intermediates of Haloalkanes, Haloalkenes, and α-Haloacids
Advances in Experimental Medicine and Biology, 2020Co-Authors: M. W. AndersAbstract:Haloalkanes, Haloalkenes, and α-Haloacids are important industrial chemicals and environmental contaminants. For example, 1,2-dibromoethane and 1,2-dibromo-3-chloropropane were formerly used as fumigants and nematocides, trichloroethylene is a common environmental contaminant, and dichloroacetate (DCA), which is produced during the chlorination of drinking water, is present in finished drinking water supplies in the U.S. Many haloalkanes, Haloalkenes, and α-haloacids are toxic, and some are rodent or suspected human carcinogens. The toxicity of these chemicals is associated with their bioactivation to reactive intermediates by the cytochromes P450 or glutathione transferases (GSTs).
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Glutathione-dependent bioactivation of haloalkanes and Haloalkenes.
Drug Metabolism Reviews, 2004Co-Authors: M. W. AndersAbstract:Haloalkanes and Haloalkenes constitute an important group of widely used chemicals that have the potential to induce toxicity and cancer. The toxicity of haloalkanes and Haloalkenes may be associated with cytochromes P450− or glutathione transferase‐dependent bioactivation. This review is concerned with the glutathione− and glutathione transferase‐dependent bioactivation of dihalomethanes, 1,2‐dihaloalkanes, and Haloalkenes. Dihalomethanes, e.g., dichloromethane, and 1,2‐dihaloethanes, e.g., 1,2‐dichloroethane and 1,2‐dibromoethane, undergo glutathione transferase‐catalyzed bioactivation to give S‐(halomethyl)glutathione or glutathione episulfonium ions, respectively, as reactive intermediates. Haloalkenes, e.g., trichloroethene, hexachlorobutadiene, chlorotrifluoroethene, and tetrafluoroethene, undergo cysteine conjugate β‐lyase‐dependent bioactivation to thioacylating intermediates, including thioacyl halides, thioketenes, and 2,2,3‐trihalothiiranes. With all of these compounds, the formation of reactiv...
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Acivicin-induced alterations in renal and hepatic glutathione concentrations and in γ-glutamyltransferase activities
Biochemical Pharmacology, 2004Co-Authors: Hoffman B. M. Lantum, Ramaswamy A. Iyer, M. W. AndersAbstract:Abstract γ-Glutamyltransferase (γ-GT) catalyzes the hydrolysis of glutathione, glutathione S-conjugates, and γ-substituted l -glutamate derivatives. Acivicin is an irreversible inhibitor of γ-GT that has been used to study the role of γ-GT in glutathione homeostasis and glutathione-dependent bioactivation reactions. The present studies were undertaken because of reported conflicting effects of acivicin on the nephrotoxicity of some Haloalkenes that undergo glutathione-dependent bioactivation. The objective of this study was to test the hypothesis that acivicin may alter renal glutathione concentrations; acivicin-induced changes in renal glutathione concentrations may alter the susceptibility of the kidney to the nephrotoxic effects of Haloalkenes. Hence, diurnal and acivicin-induced changes in renal and hepatic glutathione concentrations along with renal and hepatic γ-GT activities were investigated. The previously observed diurnal variations in hepatic glutathione concentrations in fed rats were confirmed, but no diurnal variations were observed in renal glutathione concentrations or in renal or hepatic γ-GT activities. Renal and hepatic glutathione concentrations and γ-GT activities were measured in tissue homogenates from rats given 0, 0.1, or 0.2 mmol acivicin/kg (i.p.) and killed 0, 2, 4, 8, 12, or 24 hr later. Renal glutathione concentrations were increased above control values in acivicin-treated rats, whereas acivicin had no effect on hepatic glutathione concentrations. Renal γ-GT activities decreased within 2 hr after giving acivicin and remained decreased for 24 hr. Acivicin had no effect on hepatic γ-GT activities, except at 24 hr after treatment when values in acivicin-treated rats were elevated compared with controls. Although the present studies do not afford an explanation of the mechanism whereby acivicin increases the nephrotoxicity of some Haloalkenes, they do indicate that acivicin is not a reliable probe to investigate the role of γ-GT in Haloalkene-induced nephrotoxicity.
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Glutathione-dependent bioactivation of haloalkanes and Haloalkenes.
Drug metabolism reviews, 2004Co-Authors: M. W. AndersAbstract:Haloalkanes and Haloalkenes constitute an important group of widely used chemicals that have the potential to induce toxicity and cancer. The toxicity of haloalkanes and Haloalkenes may be associated with cytochromes P450- or glutathione transferase-dependent bioactivation. This review is concerned with the glutathione- and glutathione transferase-dependent bioactivation of dihalomethanes, 1,2-dihaloalkanes, and Haloalkenes. Dihalomethanes, e.g., dichloromethane, and 1,2-dihaloethanes, e.g., 1,2-dichloroethane and 1,2-dibromoethane, undergo glutathione transferase-catalyzed bioactivation to give S-(halomethyl)glutathione or glutathione episulfonium ions, respectively, as reactive intermediates. Haloalkenes, e.g., trichloroethene, hexachlorobutadiene, chlorotrifluoroethene, and tetrafluoroethene, undergo cysteine conjugate beta-lyase-dependent bioactivation to thioacylating intermediates, including thioacyl halides, thioketenes, and 2,2,3-trihalothiiranes. With all of these compounds, the formation of reactive intermediates is associated with their observed toxicity.
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Computational and experimental studies on the distribution of addition and substitution products of the microsomal glutathione transferase 1-catalyzed conjugation of glutathione with fluoroalkenes.
Chemical Research in Toxicology, 2003Co-Authors: Larry J. Jolivette, M. W. AndersAbstract:The glutathione transferase-catalyzed reaction of glutathione with Haloalkenes results in the formation of addition or substitution products or both. Glutathione conjugates of Haloalkenes may be metabolized and excreted at different rates, may follow different metabolic pathways, and may exhibit different toxicities. Microsomal glutathione transferase 1 (MGST1)-catalyzed conjugation of chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene results in differing proportions of addition and substitution products. The aim of the present study was to develop a computational model to predict the outcome of the MGST1-catalyzed reaction of glutathione with Haloalkenes. An ab initio computational study of the reaction of ethanethiolate, a surrogate for glutathione, with the chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene was conducted. An empirical study was also conducted to quantify the distribution of addition and substitut...
