Koudelakova T

References (15)

Title : Balancing the stability-activity trade-off by fine-tuning dehalogenase access tunnels - Liskova_2015_ChemCatChem_7_648
Author(s) : Liskova V , Bednar D , Prudnikova T , Rezacova P , Koudelakova T , Sebestova E , Kuta-Smatanova I , Brezovsky J , Chaloupkova R , Damborsky J
Ref : ChemCatChem , 7 :648 , 2015
Abstract : A variant of the haloalkane dehalogenase DhaA with greatly enhanced stability and tolerance of organic solvents but reduced activity was created by mutating four residues in the access tunnel. To create a stabilized enzyme with superior catalytic activity, two of the four originally modified residues were randomized. The resulting mutant F176G exhibited 10- and 32-times enhanced activity towards 1,2-dibromoethane in buffer and 40% (v/v) DMSO, respectively, while retaining high stability. Structural and molecular dynamics analyses showed that the new variant exhibited superior activity because the F176G mutation increased the radius of the tunnel's mouth and the mobility of alpha-helices lining the tunnel. The new variant's tunnel was open in 48 % of trajectories, compared to 58 % for the wild-type, but only 0.02 % for the original four-point variant. Delicate balance between activity and stability of enzymes can be manipulated by fine-tuning the diameter and dynamics of their access tunnels.
ESTHER : Liskova_2015_ChemCatChem_7_648
PubMedSearch : Liskova_2015_ChemCatChem_7_648
Gene_locus related to this paper: rhoso-halo1

Title : Dynamics and hydration explain failed functional transformation in dehalogenase design - Sykora_2014_Nat.Chem.Biol_10_428
Author(s) : Sykora J , Brezovsky J , Koudelakova T , Lahoda M , Fortova A , Chernovets T , Chaloupkova R , Stepankova V , Prokop Z , Smatanova IK , Hof M , Damborsky J
Ref : Nat Chemical Biology , 10 :428 , 2014
Abstract : We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.
ESTHER : Sykora_2014_Nat.Chem.Biol_10_428
PubMedSearch : Sykora_2014_Nat.Chem.Biol_10_428
PubMedID: 24727901
Gene_locus related to this paper: rhoso-halo1

Title : Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites - Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
Author(s) : Chaloupkova R , Prudnikova T , Rezacova P , Prokop Z , Koudelakova T , Daniel L , Brezovsky J , Ikeda-Ohtsubo W4 , Sato Y , Kuty M , Nagata Y , Kuta Smatanova I , Damborsky J
Ref : Acta Crystallographica D Biol Crystallogr , 70 :1884 , 2014
Abstract : 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.
ESTHER : Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
PubMedSearch : Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
PubMedID: 25004965
Gene_locus related to this paper: brael-e2rv62

Title : Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel - Koudelakova_2013_Angew.Chem.Int.Ed.Engl_52_1959
Author(s) : Koudelakova T , Chaloupkova R , Brezovsky J , Prokop Z , Sebestova E , Hesseler M , Khabiri M , Plevaka M , Kulik D , Kuta Smatanova I , Rezacova P , Ettrich R , Bornscheuer UT , Damborsky J
Ref : Angew Chem Int Ed Engl , 52 :1959 , 2013
Abstract : Mutations targeting as few as four residues lining the access tunnel extended the half-life of an enzyme in 40% dimethyl sulfoxide from minutes to weeks and increased its melting temperature by 190C. Protein crystallography and molecular dynamics revealed that the tunnel residue packing is a key determinant of protein stability and the active-site accessibility for cosolvent molecules (red dots).
ESTHER : Koudelakova_2013_Angew.Chem.Int.Ed.Engl_52_1959
PubMedSearch : Koudelakova_2013_Angew.Chem.Int.Ed.Engl_52_1959
PubMedID: 23303607
Gene_locus related to this paper: rhoso-halo1

Title : Haloalkane dehalogenases: Biotechnological applications - Koudelakova_2013_Biotechnol.J_8_32
Author(s) : Koudelakova T , Bidmanova S , Dvorak P , Pavelka A , Chaloupkova R , Prokop Z , Damborsky J
Ref : Biotechnol J , 8 :32 , 2013
Abstract : Haloalkane dehalogenases (EC, HLDs) are alpha/beta-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solutions.
ESTHER : Koudelakova_2013_Biotechnol.J_8_32
PubMedSearch : Koudelakova_2013_Biotechnol.J_8_32
PubMedID: 22965918

Title : DspA from Strongylocentrotus purpuratus: The first biochemically characterized haloalkane dehalogenase of non-microbial origin - Fortova_2013_Biochimie_95_2091
Author(s) : Fortova A , Sebestova E , Stepankova V , Koudelakova T , Palkova L , Damborsky J , Chaloupkova R
Ref : Biochimie , 95 :2091 , 2013
Abstract : Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbon-halogen 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 beta-brominated alkanes. Moreover, we identified other putative haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.
ESTHER : Fortova_2013_Biochimie_95_2091
PubMedSearch : Fortova_2013_Biochimie_95_2091
PubMedID: 23939220
Gene_locus related to this paper: strpu-h3hrw2

Title : Strategies for Stabilization of Enzymes in Organic Solvents - Stepankova_2013_ACS.Catal_3_2823
Author(s) : Stepankova V , Bidmanova S , Koudelakova T , Prokop Z , Chaloupkova R , Damborsky J
Ref : ACS Catal , 3 :2823 , 2013
Abstract : One of the major barriers to the use of enzymes in industrial biotechnology is their insufficient stability under processing conditions. The use of organic solvent systems instead of aqueous media for enzymatic reactions offers numerous advantages, such as increased solubility of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcohols become thermodynamically favorable. However, organic solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of organic solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile organic solvents by ionic liquids and deep eutectic solvents.
ESTHER : Stepankova_2013_ACS.Catal_3_2823
PubMedSearch : Stepankova_2013_ACS.Catal_3_2823

Title : Biochemical characterization of a novel haloalkane dehalogenase from a cold-adapted bacterium - Drienovska_2012_Appl.Environ.Microbiol_78_4995
Author(s) : Drienovska I , Chovancova E , Koudelakova T , Damborsky J , Chaloupkova R
Ref : Applied Environmental Microbiology , 78 :4995 , 2012
Abstract : A haloalkane dehalogenase, DpcA, from Psychrobacter cryohalolentis K5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.
ESTHER : Drienovska_2012_Appl.Environ.Microbiol_78_4995
PubMedSearch : Drienovska_2012_Appl.Environ.Microbiol_78_4995
PubMedID: 22582053
Gene_locus related to this paper: psyck-q1qbb9

Title : Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58 - Hasan_2011_Appl.Environ.Microbiol_77_1881
Author(s) : Hasan K , Fortova A , Koudelakova T , Chaloupkova R , Ishitsuka M , Nagata Y , Damborsky J , Prokop Z
Ref : Applied Environmental Microbiology , 77 :1881 , 2011
Abstract : 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.
ESTHER : Hasan_2011_Appl.Environ.Microbiol_77_1881
PubMedSearch : Hasan_2011_Appl.Environ.Microbiol_77_1881
PubMedID: 21193677

Title : Substrate specificity of haloalkane dehalogenases - Koudelakova_2011_Biochem.J_435_345
Author(s) : Koudelakova T , Chovancova E , Brezovsky J , Monincova M , Fortova A , Jarkovsky J , Damborsky J
Ref : Biochemical Journal , 435 :345 , 2011
Abstract : An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
ESTHER : Koudelakova_2011_Biochem.J_435_345
PubMedSearch : Koudelakova_2011_Biochem.J_435_345
PubMedID: 21294712
Gene_locus related to this paper: agrtu-DHAA , brael-e2rv62 , braja-dhaa , myctu-linb , myctu-Rv1833c , rhoba-DHLA , rhoso-halo1 , sphpi-linb , xanau-halo1

Title : Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels - Stsiapanava_2010_Acta.Crystallogr.D.Biol.Crystallogr_66_962
Author(s) : Stsiapanava A , Dohnalek J , Gavira JA , Kuty M , Koudelakova T , Damborsky J , Kuta Smatanova I
Ref : Acta Crystallographica D Biol Crystallogr , 66 :962 , 2010
Abstract : The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.
ESTHER : Stsiapanava_2010_Acta.Crystallogr.D.Biol.Crystallogr_66_962
PubMedSearch : Stsiapanava_2010_Acta.Crystallogr.D.Biol.Crystallogr_66_962
PubMedID: 20823547
Gene_locus related to this paper: rhoso-halo1

Title : Enantioselectivity of haloalkane dehalogenases and its modulation by surface loop engineering -
Author(s) : Prokop Z , Sato Y , Brezovsky J , Mozga T , Chaloupkova R , Koudelakova T , Jerabek P , Stepankova V , Natsume R , van Leeuwen JG , Janssen DB , Florian J , Nagata Y , Senda T , Damborsky J
Ref : Angew Chem Int Ed Engl , 49 :6111 , 2010
PubMedID: 20645368
Gene_locus related to this paper: braja-dhaa

Title : Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations - Klvana_2009_J.Mol.Biol_392_1339
Author(s) : Klvana M , Pavlova M , Koudelakova T , Chaloupkova R , Dvorak P , Prokop Z , Stsiapanava A , Kuty M , Kuta-Smatanova I , Dohnalek J , Kulhanek P , Wade RC , Damborsky J
Ref : Journal of Molecular Biology , 392 :1339 , 2009
Abstract : Eight mutants of the DhaA haloalkane dehalogenase carrying mutations at the residues lining two tunnels, previously observed by protein X-ray crystallography, were constructed and biochemically characterized. The mutants showed distinct catalytic efficiencies with the halogenated substrate 1,2,3-trichloropropane. Release pathways for the two dehalogenation products, 2,3-dichloropropane-1-ol and the chloride ion, and exchange pathways for water molecules, were studied using classical and random acceleration molecular dynamics simulations. Five different pathways, denoted p1, p2a, p2b, p2c, and p3, were identified. The individual pathways showed differing selectivity for the products: the chloride ion releases solely through p1, whereas the alcohol releases through all five pathways. Water molecules play a crucial role for release of both products by breakage of their hydrogen-bonding interactions with the active-site residues and shielding the charged chloride ion during its passage through a hydrophobic tunnel. Exchange of the chloride ions, the alcohol product, and the waters between the buried active site and the bulk solvent can be realized by three different mechanisms: (i) passage through a permanent tunnel, (ii) passage through a transient tunnel, and (iii) migration through a protein matrix. We demonstrate that the accessibility of the pathways and the mechanisms of ligand exchange were modified by mutations. Insertion of bulky aromatic residues in the tunnel corresponding to pathway p1 leads to reduced accessibility to the ligands and a change in mechanism of opening from permanent to transient. We propose that engineering the accessibility of tunnels and the mechanisms of ligand exchange is a powerful strategy for modification of the functional properties of enzymes with buried active sites.
ESTHER : Klvana_2009_J.Mol.Biol_392_1339
PubMedSearch : Klvana_2009_J.Mol.Biol_392_1339
PubMedID: 19577578
Gene_locus related to this paper: rhoso-halo1

Title : Biochemical characterization of haloalkane dehalogenases DrbA and DmbC, Representatives of a Novel Subfamily - Jesenska_2009_Appl.Environ.Microbiol_75_5157
Author(s) : Jesenska A , Monincova M , Koudelakova T , Hasan K , Chaloupkova R , Prokop Z , Geerlof A , Damborsky J
Ref : Applied Environmental Microbiology , 75 :5157 , 2009
Abstract : This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.
ESTHER : Jesenska_2009_Appl.Environ.Microbiol_75_5157
PubMedSearch : Jesenska_2009_Appl.Environ.Microbiol_75_5157
PubMedID: 19502442
Gene_locus related to this paper: myctu-Rv1833c

Title : Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis - Stsiapanava_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_137
Author(s) : Stsiapanava A , Koudelakova T , Lapkouski M , Pavlova M , Damborsky J , Smatanova IK
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 64 :137 , 2008
Abstract : The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.
ESTHER : Stsiapanava_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_137
PubMedSearch : Stsiapanava_2008_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_64_137
PubMedID: 18259069
Gene_locus related to this paper: rhoso-halo1