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Dick B. Janssen - One of the best experts on this subject based on the ideXlab platform.
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Genetic Adaptation of Bacteria to Halogenated Aliphatic Compounds
2013Co-Authors: Dick B. Janssen, Jan R. Van Der Ploeg, Frens PriesAbstract:The bacterial degradation and detoxification of chlorinated xenobiotic compounds requires the production of enzymes that are capable of recognizing and converting compounds which do not occur at significant concentrations in nature. We have studied the catabolic route of 1,2-dichloroethane as an example of a pathway for the conversion of such a synthetic compound. In strains of Xanthobacter and Ancylobacter that have been isolated on 1,2-dichloroethane, the first catabolic step is catalyzed by a hydrolytic Haloalkane dehalogenase. The enzyme converts 1,2-dichloroethane to 2-chloroethanol but is also active with many other environmentally important Haloalkanes such as methylchloride, methylbromide, 1,2-dibromoethane, epichlorohydrin, and 1,3-dichloropropene. Further degradation of 2-chloroethanol proceeds by oxidation to the carboxylic acid and dehalogenation to glycolate. The aldehyde dehydrogenase prevents toxicity of the reactive chloroacetaldehyde that is formed as an intermediate and is necessary for establishing a functional 2-chloroethanol degradative pathway in a strain that is not capable of growth on this compound.- Environ Health Perspec
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Haloalkane dehalogenase catalysed desymmetrisation and tandem kinetic resolution for the preparation of chiral haloalcohols
Tetrahedron, 2012Co-Authors: Alja Westerbeek, Jan G. E. Van Leeuwen, Ben L. Feringa, Wiktor Szymanski, Dick B. JanssenAbstract:Six different bacterial Haloalkane dehalogenases were recombinantly produced in Escherichia coli, purified, and used to catalyse the conversion of prochiral short-chain diHaloalkanes and a meso diHaloalkane, yielding enantioenriched haloalcohols. A two-reaction one-enzyme process was established in which the desymmetrisation of a diHaloalkane is followed by kinetic resolution of the chiral haloalcohol that is produced in the first step. In case of 1,3-dibromo-2-methylpropane and 1,3-dibromo-2-phenylpropane, an increase of the enantiomeric excess of the respective haloalcohol was observed in time, leading to ee values of >97%, with analytical yields of 24 and 52%, respectively. The results show that Haloalkane dehalogenases can be used for the production of highly enantioenriched haloalcohols and that in some cases product enantiopurity can be improved by allowing a two-step one-enzyme tandem reaction.
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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, René 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.
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biocatalysis by dehalogenating enzymes
Advances in Applied Microbiology, 2007Co-Authors: Dick B. JanssenAbstract:Publisher Summary This chapter discusses dehalogenase enzymes and its types. The chapter emphasizes on the microbial origin and distribution of these enzymes, their biochemical properties, and their engineering and use in biocatalysis. Dehalogenases comprise a diverse group of enzymes belonging to different phylogenetic and mechanistic classes. They have biotechnological potential because of their high-catalytic power in the cleavage of carbon–halogen bonds of toxic environmental pollutants. Dehalogenases appear with very different structural folds, reaction types, and catalytic mechanisms. For example, several reductive dehalogenases that replace a chlorine substituent by a hydrogen possess a corrinoid cofactor typical for an electron transfer mechanism, whereas Haloalkane dehalogenases belong to the α/β-hydrolase fold family, a group of proteins of which most members catalyze hydrolytic reactions via a covalent alkyl-enzyme intermediate, reminiscent to the acyl-enzyme intermediate of classical serine proteases. More recently, dehalogenases have been further explored for enantioselective conversion of other halocarboxylic acids and their esters and Haloalkanes, as well as for their applicability in recycling and detoxifying trichloropropane.
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catalytic mechanism of the Haloalkane dehalogenase linb from sphingomonas paucimobilis ut26
Journal of Biological Chemistry, 2003Co-Authors: Zbyněk Prokop, Marta Monincova, Yuji Nagata, Martin Klvaňa, Radka Chaloupkova, Dick B. JanssenAbstract:Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the Haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of Haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the Haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.
Yuji Nagata - One of the best experts on this subject based on the ideXlab platform.
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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, Yuji Nagata, Radka Chaloupkova, Jiri Damborsky, 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, Fabiana Lica Imai, Yuji Nagata, Masahiko Okai, Jun Ohtsuka, 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, Yuji Nagata, Radka Chaloupkova, Jiri Damborsky, 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 x ray analysis of a novel Haloalkane dehalogenase dbea from bradyrhizobium elkani usda94
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009Co-Authors: Tatyana Prudnikova, Michal Kutý, Jiri Brynda, Pavlina Rezacova, Yuji Nagata, Tomáš Mozga, Radka Chaloupkova, Yukari Sato, Ivana Kuta SmatanovaAbstract:A novel enzyme, DbeA, belonging to the Haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P212121, while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2 A resolution for both DbeA and DbeA1 crystals.
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weak activity of Haloalkane dehalogenase linb with 1 2 3 trichloropropane revealed by x ray crystallography and microcalorimetry
Applied and Environmental Microbiology, 2007Co-Authors: Marta Monincova, Jitka Vevodova, Yuji Nagata, Zbynek Prokop, Jiri DamborskyAbstract: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.
Radka Chaloupkova - One of the best experts on this subject based on the ideXlab platform.
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deciphering the structural basis of high thermostability of dehalogenase from psychrophilic bacterium marinobacter sp elb17
Microorganisms, 2019Co-Authors: Lukas Chrast, Ivana Kuta Smatanova, Tatyana Prudnikova, Lukas Daniel, Jan Brezovský, Radka Chaloupkova, Katsiaryna Tratsiak, Joan Planasiglesias, David Bednar, Jiri DamborskyAbstract:Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new Haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type Haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 A resolution. DmxA structure contains several features distinct from known members of Haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.
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structural and functional analysis of a novel Haloalkane dehalogenase with two halide binding sites
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Radka Chaloupkova, Tatyana Prudnikova, Lukas Daniel, Wakako Ikedaohtsubo, Pavlina Rezacova, Jan Brezovský, Tana Koudelakova, Zbynek Prokop, Yukari SatoAbstract:The crystal structure of the novel Haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized Haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
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release of halide ions from the buried active site of the Haloalkane dehalogenase linb revealed by stopped flow fluorescence analysis and free energy calculations
Journal of Physical Chemistry B, 2013Co-Authors: Jana Hladilkova, Radka Chaloupkova, Zbynek Prokop, Jiri Damborsky, Pavel JungwirthAbstract:Release of halide ions is an essential step of the catalytic cycle of Haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the Haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations, we show that the anion binding is ion-specific and follows the ordering I– > Br– > Cl–. We also address the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we show that the anionic ordering does not change with the switch of the protonation state. We also demonstrate that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na+ is strongly repelled from the active site containing the doubly protonated His272 residue. Ou...
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dspa from strongylocentrotus purpuratus the first biochemically characterized Haloalkane dehalogenase of non microbial origin
Biochimie, 2013Co-Authors: Andrea Fortova, Eva Sebestova, Lenka Palkova, Tana Koudelakova, Veronika Stepankova, Jiri Damborsky, Radka ChaloupkovaAbstract:Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbonehalogen bond in halogenated compounds. Here we report the first biochemically characterized non-microbial Haloalkane dehalogenase DspA from Strongylocentrotus purpuratus. The enzyme shows a preference for terminally brominated hydrocarbons and enantioselectivity towards b-brominated alkanes. Moreover, we identified other putative Haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.
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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, Yuji Nagata, Radka Chaloupkova, Jiri Damborsky, 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.
Jiri Damborsky - One of the best experts on this subject based on the ideXlab platform.
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deciphering the structural basis of high thermostability of dehalogenase from psychrophilic bacterium marinobacter sp elb17
Microorganisms, 2019Co-Authors: Lukas Chrast, Ivana Kuta Smatanova, Tatyana Prudnikova, Lukas Daniel, Jan Brezovský, Radka Chaloupkova, Katsiaryna Tratsiak, Joan Planasiglesias, David Bednar, Jiri DamborskyAbstract:Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new Haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type Haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 A resolution. DmxA structure contains several features distinct from known members of Haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.
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controlled oil water partitioning of hydrophobic substrates extending the bioanalytical applications of droplet based microfluidics
Analytical Chemistry, 2019Co-Authors: Tomas Buryska, Michal Vasina, Fabrice Gielen, Pavel Vanacek, Liisa D Van Vliet, Jan Jezek, Zdenek Pilat, Pavel Zemanek, Jiri DamborskyAbstract:Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic Haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the Haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 degrees C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.
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Poor calibration based on slope versus a better quality calibration based on amplitudes in the case of a complex reaction kinetics.
2018Co-Authors: Stanislav Mazurenko, Sarka Bidmanova, Jiri Damborsky, Marketa Kotlanova, Zbynek ProkopAbstract:(A) The response of a biodevice based on experiments using 0.8 mg of Haloalkane dehalogenase LinB and different concentrations of environmental pollutant 1-chlorohexane; (B) calibration points for slopes and amplitudes.
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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, Ondrej Vavra, Petra Babkova, 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.
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Enzyme-Based Test Strips for Visual or Photographic Detection and Quantitation of Gaseous Sulfur Mustard
2016Co-Authors: Sarka Bidmanova, Zbynek Prokop, Jiri Damborsky, Mark-steven Steiner, Martin Stepan, Kamila Vymazalova, Michael A. Gruber, Axel Duerkop, Otto S. WolfbeisAbstract:Sulfur mustard is a chemical agent of high military and terroristic significance. No effective antidote exists, and sulfur mustard can be fairly easily produced in large quantity. Rapid field testing of sulfur mustard is highly desirable. Existing analytical devices for its detection are available but can suffer from low selectivity, laborious sample preparation, and/or the need for complex instrumentation. We describe a new kind of test strip for rapid detection of gaseous sulfur mustard that is based on its degradation by the enzyme Haloalkane dehalogenase that is accompanied by a change of local pH. This change can be detected using pH indicators contained in the strips whose color changes from blue-green to yellow within 10 min. In addition to visual read-out, we also demonstrate quantitative reflectometric readout by using a conventional digital camera based on red-green-blue data acquisition. Organic Haloalkanes, such as 1,2-dichloroethane, have a negligible interfering effect. The visual limit of detection is 20 μg/L, and the one for red-green-blue read-out is as low as 3 μg/L. The assays have good reproducibility ±6% and ±2% for interday assays and intraday assays, respectively. The strips can be stored for at least 6 months without loss of function. They are disposable and can be produced fairly rapidly and at low costs. Hence, they represent a promising tool for in-field detection of sulfur mustard
Zbynek Prokop - One of the best experts on this subject based on the ideXlab platform.
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Poor calibration based on slope versus a better quality calibration based on amplitudes in the case of a complex reaction kinetics.
2018Co-Authors: Stanislav Mazurenko, Sarka Bidmanova, Jiri Damborsky, Marketa Kotlanova, Zbynek ProkopAbstract:(A) The response of a biodevice based on experiments using 0.8 mg of Haloalkane dehalogenase LinB and different concentrations of environmental pollutant 1-chlorohexane; (B) calibration points for slopes and amplitudes.
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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, Ondrej Vavra, Petra Babkova, 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.
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Enzyme-Based Test Strips for Visual or Photographic Detection and Quantitation of Gaseous Sulfur Mustard
2016Co-Authors: Sarka Bidmanova, Zbynek Prokop, Jiri Damborsky, Mark-steven Steiner, Martin Stepan, Kamila Vymazalova, Michael A. Gruber, Axel Duerkop, Otto S. WolfbeisAbstract:Sulfur mustard is a chemical agent of high military and terroristic significance. No effective antidote exists, and sulfur mustard can be fairly easily produced in large quantity. Rapid field testing of sulfur mustard is highly desirable. Existing analytical devices for its detection are available but can suffer from low selectivity, laborious sample preparation, and/or the need for complex instrumentation. We describe a new kind of test strip for rapid detection of gaseous sulfur mustard that is based on its degradation by the enzyme Haloalkane dehalogenase that is accompanied by a change of local pH. This change can be detected using pH indicators contained in the strips whose color changes from blue-green to yellow within 10 min. In addition to visual read-out, we also demonstrate quantitative reflectometric readout by using a conventional digital camera based on red-green-blue data acquisition. Organic Haloalkanes, such as 1,2-dichloroethane, have a negligible interfering effect. The visual limit of detection is 20 μg/L, and the one for red-green-blue read-out is as low as 3 μg/L. The assays have good reproducibility ±6% and ±2% for interday assays and intraday assays, respectively. The strips can be stored for at least 6 months without loss of function. They are disposable and can be produced fairly rapidly and at low costs. Hence, they represent a promising tool for in-field detection of sulfur mustard
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Mechanism-Based Discovery of Novel Substrates of Haloalkane Dehalogenases Using in Silico Screening
2015Co-Authors: Lukas Daniel, Tomas Buryska, Zbynek Prokop, Jiri Damborsky, Jan BrezovskýAbstract:Substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of novel substrates of enzymes with a known reaction mechanism. The method was developed for a model system of biotechnologically relevant enzymes, Haloalkane dehalogenases. On the basis of the parametrization of six different Haloalkane dehalogenases with 30 halogenated substrates, mechanism-based geometric criteria for reactivity approximation were defined. These criteria were subsequently applied to the previously experimentally uncharacterized Haloalkane dehalogenase DmmA. The enzyme was computationally screened against 41,366 compounds, yielding 548 structurally unique compounds as potential substrates. Eight out of 16 experimentally tested top-ranking compounds were active with DmmA, indicating a 50% success rate for the prediction of substrates. The remaining eight compounds were able to bind to the active site and inhibit enzymatic activity. These results confirmed good applicability of the method for prioritizing active compoundstrue substrates and bindersfor experimental testing. All validated substrates were large compounds often containing polyaromatic moieties, which have never before been considered as potential substrates for this enzyme family. Whereas four of these novel substrates were specific to DmmA, two substrates showed activity with three other tested Haloalkane dehalogenases, i.e., DhaA, DbjA, and LinB. Additional validation of the developed screening strategy with the data set of over 200 known substrates of Candida antarctica lipase B confirmed its applicability for the identification of novel substrates of other biotechnologically relevant enzymes with an available tertiary structure and known reaction mechanism
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structural and functional analysis of a novel Haloalkane dehalogenase with two halide binding sites
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Radka Chaloupkova, Tatyana Prudnikova, Lukas Daniel, Wakako Ikedaohtsubo, Pavlina Rezacova, Jan Brezovský, Tana Koudelakova, Zbynek Prokop, Yukari SatoAbstract:The crystal structure of the novel Haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized Haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
