Damborsky J

General

Full name : Damborsky Jiri

First name : Jiri

Mail : Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5\/A13, 625 00 Brno

Zip Code :

City :

Country : Czech Republic

Email : jiri@chemi.muni.cz

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References (140)

Title : Exploring new galaxies: Perspectives on the discovery of novel PET-degrading enzymes - Mican_2024_Appl.Catal.B.Environmental_342_123404
Author(s) : Mican J , Jaradat DMM , Liu W , Weber G , Mazurenko S , Bornscheuer UT , Damborsky J , Wei R , Bednar D
Ref : Applied Catalysis B: Environmental , 342 :123404 , 2024
Abstract : Polyethylene terephthalate (PET) is a widely used polyester due to its beneficial material properties and low cost. However, PET contributes significantly to the growing problem of plastic waste pollution. Enzymatic PET recycling has emerged as a promising alternative to conventional mechanical and chemical recycling methods. While many PET hydrolases belonging to the a/-hydrolase fold superfamily have been discovered, the wild-type enzymes obtained from natural sources are not optimal for industrial conditions and need to be optimized through rational design or directed evolution to improve their efficiency and stability. This Perspective summarizes case studies of engineered PET hydrolases and proposes a workflow that tightly integrates a variety of in silico and high-throughput approaches for biochemical and structural characterization to accelerate the discovery of PET-degrading enzymes, also with novel structural scaffolds. These biocatalysts could be candidates for developing further innovative plastic recycling techniques.
ESTHER : Mican_2024_Appl.Catal.B.Environmental_342_123404
PubMedSearch : Mican_2024_Appl.Catal.B.Environmental_342_123404
PubMedID:

Title : Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyse the Mycotoxin Zearalenone - Fruhauf_2024_ACS.Catal_14_3392
Author(s) : Fruhauf S , Puhringer D , Thamhesl M , Fajtl P , Kunz-Vekiru E , Hobartner-Gussl A , Schatzmayr G , Adam G , Damborsky J , Djinovic-Carugo K , Prokop Z , Moll WD
Ref : ACS Catal , 14 :3392 , 2024
Abstract : Zearalenone (ZEN) is a mycoestrogenic polyketide produced by Fusarium graminearum and other phytopathogenic members of the genus Fusarium. Contamination of cereals with ZEN is frequent, and hydrolytic detoxification with fungal lactonases has been explored. Here, we report the isolation of a bacterial strain, Rhodococcus erythropolis PFA D81, with ZEN hydrolyzing activity, cloning of the gene encoding a/ hydrolase ZenA encoded on the linear megaplasmid pSFRL1, and biochemical characterization of nine homologues. Furthermore, we report site-directed mutagenesis as well as structural analysis of the dimeric ZenARe of R. erythropolis and the more thermostable, tetrameric ZenAScfl of Streptomyces coelicoflavus with and without bound ligands. The X-ray crystal structures not only revealed canonical features of alpha/beta hydrolases with a cap domain including a Ser-His-Asp catalytic triad but also unusual features including an uncommon oxyanion hole motif and a peripheral, short antiparallel -sheet involved in tetramer interactions. Presteady-state kinetic analyses for ZenARe and ZenAScfl identified balanced rate-limiting steps of the reaction cycle, which can change depending on temperature. Some new bacterial ZEN lactonases have lower KM and higher kcat than the known fungal ZEN lactonases and may lend themselves to enzyme technology development for the degradation of ZEN in feed or food.
ESTHER : Fruhauf_2024_ACS.Catal_14_3392
PubMedSearch : Fruhauf_2024_ACS.Catal_14_3392
PubMedID: 38449531
Gene_locus related to this paper: 9actn-ZenA , 9noca-a0aa46n777

Title : Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum - Snajdarova_2023_Acta.Crystallogr.D.Struct.Biol__
Author(s) : Snajdarova K , Marques SM , Damborsky J , Bednar D , Marek M
Ref : Acta Crystallographica D Struct Biol , : , 2023
Abstract : Haloalkane dehalogenases (HLDs) are a family of alpha/beta-hydrolase fold enzymes that employ S(N)2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85A resolution. The structures show a canonical alphabetaalpha-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.
ESTHER : Snajdarova_2023_Acta.Crystallogr.D.Struct.Biol__
PubMedSearch : Snajdarova_2023_Acta.Crystallogr.D.Struct.Biol__
PubMedID: 37860958

Title : Deciphering Enzyme Mechanisms with Engineered Ancestors and Substrate Analogues - Gao_2023_ChemCatChem_15_e202300745
Author(s) : Gao T , Damborsky J , Janin YL , Marek M
Ref : ChemCatChem , 15 :e202300745 , 2023
Abstract : Environmentally friendly industrial and biotech processes greatly benefit from enzyme-based technologies. Their use is often possible only when the enzyme-catalytic mechanism is thoroughly known. Thus, atomic-level knowledge of a Michaelis enzyme-substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme-catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X-ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme-substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla-type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non-oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProt ASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability.
ESTHER : Gao_2023_ChemCatChem_15_e202300745
PubMedSearch : Gao_2023_ChemCatChem_15_e202300745
PubMedID:
Gene_locus related to this paper: renre-luc

Title : Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and X-ray crystallography - Chmelova_2023_Protein.Sci__e4751
Author(s) : Chmelova K , Gao T , Polak M , Schenkmayerova A , Croll TI , Shaikh TR , Skarupova J , Chaloupkova R , Diederichs K , Read RJ , Damborsky J , Novacek J , Marek M
Ref : Protein Science , :e4751 , 2023
Abstract : Haloalkane dehalogenase (HLD) enzymes employ an S(N) 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but a mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and X-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeA(deltaGG) ) that provided diffraction-quality crystals. The 3.3 A crystal structure reveals that DhmeA(deltaGG) forms a ring-like 20-mer structure with outer and inner diameter of ~200 A and ~80 A, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates. This article is protected by copyright. All rights reserved.
ESTHER : Chmelova_2023_Protein.Sci__e4751
PubMedSearch : Chmelova_2023_Protein.Sci__e4751
PubMedID: 37574754
Gene_locus related to this paper: halmt-DhmeAIII

Title : Study of Protein Conformational Dynamics Using Hydrogen\/Deuterium Exchange Mass Spectrometry - Uhrik_2023_Methods.Mol.Biol_2652_293
Author(s) : Uhrik L , Henek T , Planas-Iglesias J , Kucera J , Damborsky J , Marek M , Hernychova L
Ref : Methods Mol Biol , 2652 :293 , 2023
Abstract : Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.
ESTHER : Uhrik_2023_Methods.Mol.Biol_2652_293
PubMedSearch : Uhrik_2023_Methods.Mol.Biol_2652_293
PubMedID: 37093484

Title : Catalytic mechanism for Renilla-type luciferases - Schenkmayerova_2023_Nat.Catal_6_23
Author(s) : Schenkmayerova A , Toul M , Pluskal D , Baatallah R , Gagnot G , Pinto GP , Santana VT , Stuchla M , Neugebauer P , Chaiyen P , Damborsky J , Bednar D , Janin YL , Prokop Z , Marek M
Ref : Nature Catalysis , 6 :23 , 2023
Abstract : The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago, but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type catalysis through crystallographic, spectroscopic and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing molecular snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.
ESTHER : Schenkmayerova_2023_Nat.Catal_6_23
PubMedSearch : Schenkmayerova_2023_Nat.Catal_6_23
PubMedID:
Gene_locus related to this paper: 9zzzz-AncHLDRLuc2 , renre-luc

Title : Advancing Enzyme's Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning - Kunka_2023_ACS.Catal_13_12506
Author(s) : Kunka A , Marques SM , Havlasek M , Vasina M , Velatova N , Cengelova L , Kovar D , Damborsky J , Marek M , Bednar D , Prokop Z
Ref : ACS Catal , 13 :12506 , 2023
Abstract : Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.
ESTHER : Kunka_2023_ACS.Catal_13_12506
PubMedSearch : Kunka_2023_ACS.Catal_13_12506
PubMedID: 37822856
Gene_locus related to this paper: rhoso-halo1

Title : Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization - von Haugwitz_2022_ACS.Catal_12_15259
Author(s) : von Haugwitz G , Han X , Pfaff L , Li Q , Wei H , Gao J , Methling K , Ao Y , Brack Y , Jan Mican J , Feiler CG , Weiss MS , Bednar D , Palm GJ , Lalk M , Lammers M , Damborsky J , Weber G , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :15259 , 2022
Abstract : TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.
ESTHER : von Haugwitz_2022_ACS.Catal_12_15259
PubMedSearch : von Haugwitz_2022_ACS.Catal_12_15259
PubMedID: 36570084
Gene_locus related to this paper: thefu-1831

Title : CalFitter 2.0: Leveraging the power of singular value decomposition to analyse protein thermostability - Kunka_2022_Nucleic.Acids.Res__
Author(s) : Kunka A , Lacko D , Stourac J , Damborsky J , Prokop Z , Mazurenko S
Ref : Nucleic Acids Research , : , 2022
Abstract : The importance of the quantitative description of protein unfolding and aggregation for the rational design of stability or understanding the molecular basis of protein misfolding diseases is well established. Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals during unfolding. The CalFitter webserver has already proved integral to deriving invaluable energy parameters by global data analysis. Here, we introduce CalFitter 2.0, which newly incorporates singular value decomposition (SVD) of multi-wavelength spectral datasets into the global fitting pipeline. Processed time- or temperature-evolved SVD components can now be fitted together with other experimental data types. Moreover, deconvoluted basis spectra provide spectral fingerprints of relevant macrostates populated during unfolding, which greatly enriches the information gains of the CalFitter output. The SVD analysis is fully automated in a highly interactive module, providing access to the results to users without any prior knowledge of the underlying mathematics. Additionally, a novel data uploading wizard has been implemented to facilitate rapid and easy uploading of multiple datasets. Together, the newly introduced changes significantly improve the user experience, making this software a unique, robust, and interactive platform for the analysis of protein thermal denaturation data. The webserver is freely accessible at https://loschmidt.chemi.muni.cz/calfitter.
ESTHER : Kunka_2022_Nucleic.Acids.Res__
PubMedSearch : Kunka_2022_Nucleic.Acids.Res__
PubMedID: 35580052

Title : Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase - Pfaff_2022_ACS.Catalysis_12_9790
Author(s) : Pfaff L , Gao J , Li Z , Jackering A , Weber G , Mican J , Chen Y , Dong W , Han X , Feiler CG , Ao YF , Badenhorst CPS , Bednar D , Palm GJ , Lammers M , Damborsky J , Strodel B , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :9790 , 2022
Abstract : Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substratebinding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/ Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic r"cycling processes.
ESTHER : Pfaff_2022_ACS.Catalysis_12_9790
PubMedSearch : Pfaff_2022_ACS.Catalysis_12_9790
PubMedID: 35966606
Gene_locus related to this paper: 9firm-PHL7

Title : LoopGrafter: a web tool for transplanting dynamical loops for protein engineering - Planas-Iglesias_2022_Nucleic.Acids.Res__
Author(s) : Planas-Iglesias J , Opaleny F , Ulbrich P , Stourac J , Sanusi Z , Pinto GP , Schenkmayerova A , Byska J , Damborsky J , Kozlikova B , Bednar D
Ref : Nucleic Acids Research , : , 2022
Abstract : The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.
ESTHER : Planas-Iglesias_2022_Nucleic.Acids.Res__
PubMedSearch : Planas-Iglesias_2022_Nucleic.Acids.Res__
PubMedID: 35438789

Title : A catalytic mechanism for Renilla-type bioluminescence - Schenkmayerova_2022_Biorxiv__
Author(s) : Schenkmayerova A , Toul M , Pluskal D , Baatallah R , Gagnot G , Pinto GP , Santana VT , Stuchla M , Neugebauer P , Chaiyen P , Damborsky J , Bednar D , Janin YL , Prokop Z , Marek M
Ref : Biorxiv , : , 2022
Abstract : The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.
ESTHER : Schenkmayerova_2022_Biorxiv__
PubMedSearch : Schenkmayerova_2022_Biorxiv__
PubMedID:
Gene_locus related to this paper: 9zzzz-AncHLDRLuc2 , renre-luc

Title : Tools for computational design and high-throughput screening of therapeutic enzymes - Vasina_2022_Adv.Drug.Deliv.Rev_183_114143
Author(s) : Vasina M , Velecky J , Planas-Iglesias J , Marques SM , Skarupova J , Damborsky J , Bednar D , Mazurenko S , Prokop Z
Ref : Adv Drug Deliv Rev , 183 :114143 , 2022
Abstract : Therapeutic enzymes are valuable biopharmaceuticals in various biomedical applications. They have been successfully applied for fibrinolysis, cancer treatment, enzyme replacement therapies, and the treatment of rare diseases. Still, there is a permanent demand to find new or better therapeutic enzymes, which would be sufficiently soluble, stable, and active to meet specific medical needs. Here, we highlight the benefits of coupling computational approaches with high-throughput experimental technologies, which significantly accelerate the identification and engineering of catalytic therapeutic agents. New enzymes can be identified in genomic and metagenomic databases, which grow thanks to next-generation sequencing technologies exponentially. Computational design and machine learning methods are being developed to improve catalytically potent enzymes and predict their properties to guide the selection of target enzymes. High-throughput experimental pipelines, increasingly relying on microfluidics, ensure functional screening and biochemical characterization of target enzymes to reach efficient therapeutic enzymes.
ESTHER : Vasina_2022_Adv.Drug.Deliv.Rev_183_114143
PubMedSearch : Vasina_2022_Adv.Drug.Deliv.Rev_183_114143
PubMedID: 35167900

Title : Mechanism-Based Design of Efficient PET Hydrolases - Wei_2022_ACS.Catal_12_3382
Author(s) : Wei R , von Haugwitz G , Pfaff L , Mican J , Badenhorst CPS , Liu W , Weber G , Austin HP , Bednar D , Damborsky J , Bornscheuer UT
Ref : ACS Catal , 12 :3382 , 2022
Abstract : Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.
ESTHER : Wei_2022_ACS.Catal_12_3382
PubMedSearch : Wei_2022_ACS.Catal_12_3382
PubMedID: 35368328

Title : Description of Transport Tunnel in Haloalkane Dehalogenase Variant LinB D147C+L177C from Sphingobium japonicum - Iermak_2021_Catalysts_11_5
Author(s) : Iermak I , Degtjarik O , Havlickova P , Kuty M , Chaloupkova R , Damborsky J , Prudnikova T , Smatanova IK
Ref : Catalysts , 11 :5 , 2021
Abstract : The activity of enzymes with active sites buried inside their protein core highly depends on the efficient transport of substrates and products between the active site and the bulk solvent. The engineering of access tunnels in order to increase or decrease catalytic activity and specificity in a rational way is a challenging task. Here, we describe a combined experimental and computational approach to characterize the structural basis of altered activity in the haloalkane dehalogenase LinB D147C+L177C variant. While the overall protein fold is similar to the wild type enzyme and the other LinB variants, the access tunnels have been altered by introduced cysteines that were expected to form a disulfide bond. Surprisingly, the mutations have allowed several conformations of the amino acid chain in their vicinity, interfering with the structural analysis of the mutant by X-ray crystallography. The duration required for the growing of protein crystals changed from days to 1.5 years by introducing the substitutions. The haloalkane dehalogenase LinB D147C+L177C variant crystal structure was solved to 1.15 A resolution, characterized and deposited to Protein Data Bank under PDB ID 6s06
ESTHER : Iermak_2021_Catalysts_11_5
PubMedSearch : Iermak_2021_Catalysts_11_5
PubMedID:
Gene_locus related to this paper: sphpi-linb

Title : Substrate inhibition by the blockage of product release and its control by tunnel engineering - Kokkonen_2021_RSC.Chem.Biol_2_645
Author(s) : Kokkonen P , Beier A , Mazurenko S , Damborsky J , Bednar D , Prokop Z
Ref : RSC Chemical Biology , 2 :645 , 2021
Abstract : Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels.
ESTHER : Kokkonen_2021_RSC.Chem.Biol_2_645
PubMedSearch : Kokkonen_2021_RSC.Chem.Biol_2_645
PubMedID: 34458806
Gene_locus related to this paper: sphpi-linb

Title : Web-based tools for computational enzyme design - Marques_2021_Curr.Opin.Struct.Biol_69_19
Author(s) : Marques SM , Planas-Iglesias J , Damborsky J
Ref : Current Opinion in Structural Biology , 69 :19 , 2021
Abstract : Enzymes are in high demand for very diverse biotechnological applications. However, natural biocatalysts often need to be engineered for fine-tuning their properties towards the end applications, such as the activity, selectivity, stability to temperature or co-solvents, and solubility. Computational methods are increasingly used in this task, providing predictions that narrow down the space of possible mutations significantly and can enormously reduce the experimental burden. Many computational tools are available as web-based platforms, making them accessible to non-expert users. These platforms are typically user-friendly, contain walk-throughs, and do not require deep expertise and installations. Here we describe some of the most recent outstanding web-tools for enzyme engineering and formulate future perspectives in this field.
ESTHER : Marques_2021_Curr.Opin.Struct.Biol_69_19
PubMedSearch : Marques_2021_Curr.Opin.Struct.Biol_69_19
PubMedID: 33667757

Title : Engineering the protein dynamics of an ancestral luciferase - Schenkmayerova_2021_Nat.Commun_12_3616
Author(s) : Schenkmayerova A , Pinto GP , Toul M , Marek M , Hernychova L , Planas-Iglesias J , Daniel Liskova V , Pluskal D , Vasina M , Emond S , Dorr M , Chaloupkova R , Bednar D , Prokop Z , Hollfelder F , Bornscheuer UT , Damborsky J
Ref : Nat Commun , 12 :3616 , 2021
Abstract : Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein Anc(HLD-RLuc) which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of Anc(HLD-RLuc) challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.
ESTHER : Schenkmayerova_2021_Nat.Commun_12_3616
PubMedSearch : Schenkmayerova_2021_Nat.Commun_12_3616
PubMedID: 34127663
Gene_locus related to this paper: renre-luc

Title : Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers - Markova_2021_ACS.Catal_11_12864
Author(s) : Markova K , Kunka A , Chmelova K , Havlasek M , Babkova P , Marques SM , Vasina M , Planas-Iglesias J , Chaloupkova R , Bednar D , Prokop Z , Damborsky J , Marek M
Ref : ACS Catal , 11 :12864 , 2021
Abstract : The functionality of an enzyme depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric alpha/beta-hydrolase enzyme (Tm = 73.5 C; deltaTm > 23 C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain swapping occurs exclusively in vivo. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation biocatalysts.
ESTHER : Markova_2021_ACS.Catal_11_12864
PubMedSearch : Markova_2021_ACS.Catal_11_12864
PubMedID:
Gene_locus related to this paper: rhoso-halo1

Title : Structure-activity relationships of dually-acting acetylcholinesterase inhibitors derived from tacrine on N-methyl-d-Aspartate receptors - Gorecki_2021_Eur.J.Med.Chem_219_113434
Author(s) : Gorecki L , Misiachna A , Damborsky J , Dolezal R , Korabecny J , Cejkova L , Hakenova K , Chvojkova M , Karasova JZ , Prchal L , Novak M , Kolcheva M , Kortus S , Vales K , Horak M , Soukup O
Ref : Eur Journal of Medicinal Chemistry , 219 :113434 , 2021
Abstract : Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-MEOTA), is ensured via a "foot-in-the-door" open-channel blockage, and that interestingly both tacrine and 7-MEOTA are slightly more potent at the GluN1/GluN2A receptors when compared with the GluN1/GluN2B receptors. Here, we report that in a series of 30 novel tacrine derivatives, designed for assessment of structure-activity relationship, blocking efficacy differs among different compounds and receptors using electrophysiology with HEK293 cells expressing the defined types of NMDARs. Selected compounds (4 and 5) potently inhibited both GluN1/GluN2A and GluN1/GluN2B receptors; other compounds (7 and 23) more effectively inhibited the GluN1/GluN2B receptors; or the GluN1/GluN2A receptors (21 and 28). QSAR study revealed statistically significant model for the data obtained for inhibition of GluN1/Glu2B at -60 mV expressed as IC(50) values, and for relative inhibition of GluN1/Glu2A at +40 mV caused by a concentration of 100 microM. The models can be utilized for a ligand-based virtual screening to detect potential candidates for inhibition of GluN1/Glu2A and/or GluN1/Glu2B subtypes. Using in vivo experiments in rats we observed that unlike MK-801, the tested novel compounds did not induce hyperlocomotion in open field, and also did not impair prepulse inhibition of startle response, suggesting minimal induction of psychotomimetic side effects. We conclude that tacrine derivatives are promising compounds since they are centrally available subtype-specific inhibitors of the NMDARs without detrimental behavioral side-effects.
ESTHER : Gorecki_2021_Eur.J.Med.Chem_219_113434
PubMedSearch : Gorecki_2021_Eur.J.Med.Chem_219_113434
PubMedID: 33892271

Title : The tetrameric structure of the novel haloalkane dehalogenase DpaA from Paraglaciecola agarilytica NO2 - Mazur_2021_Acta.Crystallogr.D.Struct.Biol_77_347
Author(s) : Mazur A , Prudnikova T , Grinkevich P , Mesters JR , Mrazova D , Chaloupkova R , Damborsky J , Kuty M , Kolenko P , Kuta Smatanova I
Ref : Acta Crystallographica D Struct Biol , 77 :347 , 2021
Abstract : Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0A resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.
ESTHER : Mazur_2021_Acta.Crystallogr.D.Struct.Biol_77_347
PubMedSearch : Mazur_2021_Acta.Crystallogr.D.Struct.Biol_77_347
PubMedID: 33645538
Gene_locus related to this paper: 9alte-k6xnl5

Title : Promiscuous Dehalogenase Activity of the Epoxide Hydrolase CorEH from Corynebacterium sp. C12 - Schuiten_2021_ACS.Catal_11_6113
Author(s) : Schuiten ED , Badenhorst CPS , Palm GJ , Berndt L , Lammers M , Mican J , Bednar D , Damborsky J , Bornscheuer UT
Ref : ACS Catal , 11 :6113 , 2021
Abstract : Haloalkane dehalogenases and epoxide hydrolases are phylogenetically related and structurally homologous enzymes that use nucleophilic aspartate residues for an SN2 attack on their substrates. Despite their mechanistic similarities, no enzymes are known that exhibit both epoxide hydrolase and dehalogenase activity. We screened a subset of epoxide hydrolases, closely related to dehalogenases, for dehalogenase activity and found that the epoxide hydrolase CorEH from Corynebacterium sp. C12 exhibits promiscuous dehalogenase activity. Compared to the hydrolysis of epoxides like cyclohexene oxide (1.41 micromol min-1 mg-1), the dehalogenation of haloalkanes like 1-bromobutane (0.25 nmol min-1 mg-1) is about 5000-fold lower. In addition to the activity with 1-bromobutane, dehalogenase activity was detected with other substrates like 1-bromohexane, 1,2-dibromoethane, 1-iodobutane, and 1-iodohexane. This study shows that dual epoxide hydrolase and dehalogenase activity can be present in one naturally occurring protein scaffold.
ESTHER : Schuiten_2021_ACS.Catal_11_6113
PubMedSearch : Schuiten_2021_ACS.Catal_11_6113
PubMedID:
Gene_locus related to this paper: corsp-cEH

Title : Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA - Mazur_2021_Int.J.Mol.Sci_22_11992
Author(s) : Mazur A , Grinkevich P , Chaloupkova R , Havlickova P , Kascakova B , Kuty M , Damborsky J , Kuta Smatanova I , Prudnikova T
Ref : Int J Mol Sci , 22 : , 2021
Abstract : Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 A resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.
ESTHER : Mazur_2021_Int.J.Mol.Sci_22_11992
PubMedSearch : Mazur_2021_Int.J.Mol.Sci_22_11992
PubMedID: 34769421
Gene_locus related to this paper: 9zzzz-AncLinB

Title : Functional and Mechanistic Characterization of an Enzyme Family Combining Bioinformatics and High-Throughput Microfluidics - Vasina_2021_ResearchSquare__
Author(s) : Vasina M , Vanacek P , Hon J , Kovar D , Faldynova H , Kunka A , Badenhorst C , Buryska T , Mazurenko S , Bednar D , Stavros S , Bornscheuer U , deMello A , Damborsky J , Prokop Z
Ref : ResearchSquare , : , 2021
Abstract : https://www.researchsquare.com/article/rs-1027271/v1 Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data raises the need to speed up functional analysis. Herein, we present a pipeline integrating bioinformatics and microfluidics and its application for high-throughput mining of novel haloalkane dehalogenases. We employed bioinformatics to identify 2,905 putative dehalogenases and selected 45 representative enzymes, of which 24 were produced in soluble form. Droplet-based microfluidics accelerates subsequent experimental testing up to 20,000 reactions per day while achieving 1,000-fold lower protein consumption. This resulted in doubling the dehalogenation 'toolbox' characterized over three decades, yielding biocatalysts surpassing the efficiency of currently available enzymes. Combining microfluidics with modern global data analysis provided precious mechanistic information related to the high catalytic efficiency of new variants. This pipeline applied to other enzyme families can accelerate the identification of biocatalysts for industrial applications as well as the collection of high-quality data for machine learning.
ESTHER : Vasina_2021_ResearchSquare__
PubMedSearch : Vasina_2021_ResearchSquare__
PubMedID:
Gene_locus related to this paper: brabe-DbbA , strpu-DspB , sacko-DskA , capte-r7umg5 , shehh-b0tml1 , 9gamm-a6faz5 , 9arch-a0a1q9njs0 , 9noca-DrxA , sphsx-DspxA , 9caul-a0a1e4h0f2 , 9prot-a0a0f2rdt1 , 9noca-k0eup9 , triha-a0a0f9y4y5 , 9rhob-a0a0n7m2p6 , triha-a0a0f9x982 , rhile-DrgA , 9sphn-a0a2d6h3s9 , ensad-DeaA , 9delt-a0a0m4d813 , 9actn-a0a1g6re71 , 9actn-g7h6v4 , 9eury-j3a357 , 9alte-k7apw6 , 9sphn-a3wc35 , 9actn-a0a0c1uk61 , 9pseu-DathA , 9rhob-a0a2t6cbq8 , amys7-DaxA , lenae-DlaA , 9rhob-DpxA , 9actn-a0a1s1sm32 , 9gamm-DtacA , 9eury-a0a1i6hey7 , 9eury-m0il93 , 9actn-l7fa57 , chlad-b8g485 , 9bact-q6sht4 , 9gamm-a0z6e4 , 9rhob-a3sf89 , desps-q6ajw5 , mycmm-b2hjb4 , mycua-a0pum4 , phopr-Q93CH1 , mycav-t2gun3

Title : Stabilization of Haloalkane Dehalogenase Structure by Interfacial Interaction with Ionic Liquids - Shaposhnikova_2021_Crystals_11_1052
Author(s) : Shaposhnikova A , Kuty M , Chaloupkova R , Damborsky J , Smatanova IK , Minofar B , Prudnikova T
Ref : Crystals , 11 :1052 , 2021
Abstract : Ionic liquids attracted interest as green alternatives to replace conventional organic solvents in protein stability studies. They can play an important role in the stabilization of enzymes such as haloalkane dehalogenases that are used for biodegradation of warfare agents and halogenated environmental pollutants. Three-dimensional crystals of haloalkane dehalogenase variant DhaA80 (T148L+G171Q+A172V+C176F) from Rhodococcus rhodochrous NCIMB 13064 were grown and soaked with the solutions of 2-hydroxyethylammonium acetate and 1-butyl-3-methylimidazolium methyl sulfate. The objective was to study the structural basis of the interactions between the ionic liquids and the protein. The diffraction data were collected for the 1.25 A resolution for 2-hydroxyethylammonium acetate and 1.75 A resolution for 1-butyl-3-methylimidazolium methyl sulfate. The structures were used for molecular dynamics simulations to study the interactions of DhaA80 with the ionic liquids. The findings provide coherent evidence that ionic liquids strengthen both the secondary and tertiary protein structure due to extensive hydrogen bond interactions.
ESTHER : Shaposhnikova_2021_Crystals_11_1052
PubMedSearch : Shaposhnikova_2021_Crystals_11_1052
PubMedID:
Gene_locus related to this paper: rhoso-halo1

Title : Functional Annotation of an Enzyme Family by Integrated Strategy Combining Bioinformatics with Microanalytical and Microfluidic Technologies - Vanacek_2021_bioRxiv__
Author(s) : Vanacek P , Vasina M , Hon J , Kovar D , Faldynova H , Kunka A , Buryska T , Badenhorst CPS , Mazurenko S , Bednar D , Bornscheuer UT , Damborsky J , Prokop Z
Ref : Biorxiv , : , 2021
Abstract : Next-generation sequencing technologies enable doubling of the genomic databases every 2.5 years. Collected sequences represent a rich source of novel biocatalysts. However, the rate of accumulation of sequence data exceeds the rate of functional studies, calling for acceleration and miniaturization of biochemical assays. Here, we present an integrated platform employing bioinformatics, microanalytics, and microfluidics and its application for exploration of unmapped sequence space, using haloalkane dehalogenases as model enzymes. First, we employed bioinformatic analysis for identification of 2,905 putative dehalogenases and rational selection of 45 representative enzymes. Second, we expressed and experimentally characterized 24 enzymes showing sufficient solubility for microanalytical and microfluidic testing. Miniaturization increased the throughput to 20,000 reactions per day with 1000-fold lower protein consumption compared to conventional assays. A single run of the platform doubled dehalogenation toolbox of family members characterized over three decades. Importantly, the dehalogenase activities of nearly one-third of these novel biocatalysts far exceed that of most published HLDs. Two enzymes showed unusually narrow substrate specificity, never before reported for this enzyme family. The strategy is generally applicable to other enzyme families, paving the way towards the acceleration of the process of identification of novel biocatalysts for industrial applications but also for the collection of homogenous data for machine learning. The automated in silico workflow has been released as a user-friendly web-tool EnzymeMiner: https://loschmidt.chemi.muni.cz/enzymeminer/.
ESTHER : Vanacek_2021_bioRxiv__
PubMedSearch : Vanacek_2021_bioRxiv__
PubMedID:

Title : Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling - Dockalova_2020_Comput.Struct.Biotechnol.J_18_922
Author(s) : Dockalova V , Sanchez-Carnerero EM , Dunajova Z , Palao E , Slanska M , Buryska T , Damborsky J , Klan P , Prokop Z
Ref : Comput Struct Biotechnol J , 18 :922 , 2020
Abstract : Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the alpha/beta-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 M(-1) s(-1)) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 M(-1) s(-1)). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.
ESTHER : Dockalova_2020_Comput.Struct.Biotechnol.J_18_922
PubMedSearch : Dockalova_2020_Comput.Struct.Biotechnol.J_18_922
PubMedID: 32346465

Title : The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step - Kokkonen_2020_Comput.Struct.Biotechnol.J_18_805
Author(s) : Kokkonen P , Slanska M , Dockalova V , Pinto GP , Sanchez-Carnerero EM , Damborsky J , Klan P , Prokop Z , Bednar D
Ref : Comput Struct Biotechnol J , 18 :805 , 2020
Abstract : Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.
ESTHER : Kokkonen_2020_Comput.Struct.Biotechnol.J_18_805
PubMedSearch : Kokkonen_2020_Comput.Struct.Biotechnol.J_18_805
PubMedID: 32308927

Title : Engineering Protein Dynamics of Ancestral Luciferase - Schenkmayerova_2020_Chemrxiv__
Author(s) : Schenkmayerova A , Pinto GP , Toul M , Marek M , Hernychova L , Planas-Iglesias J , Liskova V , Pluskal D , Vasina M , Emond S , Dorr M , Chaloupkova R , Bednar D , Prokop Z , Hollfelder F , Bornscheuer UT , Damborsky J
Ref : Chemrxiv , : , 2020
Abstract : Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment's key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of a dynamic feature, provides a potent strategy for discovering protein modifications with globally disruptive but functionally innovative effects.
ESTHER : Schenkmayerova_2020_Chemrxiv__
PubMedSearch : Schenkmayerova_2020_Chemrxiv__
PubMedID:
Gene_locus related to this paper: renre-luc

Title : Exploration of enzyme diversity: High-throughput techniques for protein production and microscale biochemical characterization - Vasina_2020_Methods.Enzymol_643_51
Author(s) : Vasina M , Vanacek P , Damborsky J , Prokop Z
Ref : Methods Enzymol , 643 :51 , 2020
Abstract : Enzymes are being increasingly utilized for acceleration of industrially and pharmaceutically critical chemical reactions. The strong demand for finding robust and efficient biocatalysts for these applications can be satisfied via the exploration of enzyme diversity. The first strategy is to mine the natural diversity, represented by millions of sequences available in the public genomic databases, by using computational approaches. Alternatively, metagenomic libraries can be targeted experimentally or computationally to explore the natural diversity of a specific environment. The second strategy, known as directed evolution, is to generate man-made diversity in the laboratory using gene mutagenesis and screen the constructed library of mutants. The selected hits must be experimentally characterized in both strategies, which currently represent the rate-limiting step in the process of diversity exploration. The traditional techniques used for biochemical characterization are time-demanding, cost, and sample volume ineffective, and low-throughput. Therefore, the development and implementation of high-throughput experimental methods are essential for discovering novel enzymes. This chapter describes the experimental protocols employing the combination of robust production and high-throughput microscale biochemical characterization of enzyme variants. We validated its applicability against the model enzyme family of haloalkane dehalogenases. These protocols can be adapted to other enzyme families, paving the way towards the functional characterization and quick identification of novel biocatalysts.
ESTHER : Vasina_2020_Methods.Enzymol_643_51
PubMedSearch : Vasina_2020_Methods.Enzymol_643_51
PubMedID: 32896287

Title : EnzymeMiner: automated mining of soluble enzymes with diverse structures, catalytic properties and stabilities - Hon_2020_Nucleic.Acids.Res__
Author(s) : Hon J , Borko S , Stourac J , Prokop Z , Zendulka J , Bednar D , Martinek T , Damborsky J
Ref : Nucleic Acids Research , : , 2020
Abstract : Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochemical characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochemical characterization based on rational and robust analysis of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab experiments. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a soluble form in Escherichia coli. The solubility prediction employs the in-house SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step analysis, sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page. EnzymeMiner is a universal tool applicable to any enzyme family that provides an interactive and easy-to-use web interface freely available at https://loschmidt.chemi.muni.cz/enzymeminer/.
ESTHER : Hon_2020_Nucleic.Acids.Res__
PubMedSearch : Hon_2020_Nucleic.Acids.Res__
PubMedID: 32392342

Title : A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability - Chmelova_2020_Appl.Environ.Microbiol_86_
Author(s) : Chmelova K , Sebestova E , Liskova V , Beier A , Bednar D , Prokop Z , Chaloupkova R , Damborsky J
Ref : Applied Environmental Microbiology , 86 : , 2020
Abstract : Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same alpha-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of beta-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.
ESTHER : Chmelova_2020_Appl.Environ.Microbiol_86_
PubMedSearch : Chmelova_2020_Appl.Environ.Microbiol_86_
PubMedID: 32561584
Gene_locus related to this paper: sacvd-DsvA

Title : Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics - Babkova_2020_Comput.Struct.Biotechnol.J_18_1497
Author(s) : Babkova P , Dunajova Z , Chaloupkova R , Damborsky J , Bednar D , Marek M
Ref : Comput Struct Biotechnol J , 18 :1497 , 2020
Abstract : Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (DeltaT (m) up to 24 degreeC), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.
ESTHER : Babkova_2020_Comput.Struct.Biotechnol.J_18_1497
PubMedSearch : Babkova_2020_Comput.Struct.Biotechnol.J_18_1497
PubMedID: 32637047
Gene_locus related to this paper: 9bact-AncHLD3 , 9bact-AncHLD2 , 9bact-AncHLD5

Title : Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family - Marek_2020_Comput.Struct.Biotechnol.J_18_1352
Author(s) : Marek M , Chaloupkova R , Prudnikova T , Sato Y , Rezacova P , Nagata Y , Kuta Smatanova I , Damborsky J
Ref : Comput Struct Biotechnol J , 18 :1352 , 2020
Abstract : Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and beta4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (DeltaT (m) = -5 to 6 degC). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.
ESTHER : Marek_2020_Comput.Struct.Biotechnol.J_18_1352
PubMedSearch : Marek_2020_Comput.Struct.Biotechnol.J_18_1352
PubMedID: 32612758
Gene_locus related to this paper: brael-e2rv62

Title : Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst - Markova_2020_Chem.Sci_11_11162
Author(s) : Markova K , Chmelova K , Marques SM , Carpentier P , Bednar D , Damborsky J , Marek M
Ref : Chem Sci , 11 :11162 , 2020
Abstract : Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T (m) = 73.5 degreesC; deltaT (m) > 23 degreesC). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 A and 1.6 A resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the alphabetaalpha-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (-27 A) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.
ESTHER : Markova_2020_Chem.Sci_11_11162
PubMedSearch : Markova_2020_Chem.Sci_11_11162
PubMedID: 34094357
Gene_locus related to this paper: rhoso-halo1

Title : Controlled Oil\/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics - Buryska_2019_Anal.Chem_91_10008
Author(s) : Buryska T , Vasina M , Gielen F , Vanacek P , van Vliet L , Jezek J , Pilat Z , Zemanek P , Damborsky J , Hollfelder F , Prokop Z
Ref : Analytical Chemistry , 91 :10008 , 2019
Abstract : 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 65000-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 >20000 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.
ESTHER : Buryska_2019_Anal.Chem_91_10008
PubMedSearch : Buryska_2019_Anal.Chem_91_10008
PubMedID: 31240908

Title : Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts - Chaloupkova_2019_ACS.Catal_9_48103
Author(s) : Chaloupkova, R , Liskova V , Tool M , Markova K , Sebestova E , Hernychova L , Marek M , Pinto GP , Pluskal D , Waterman J , Prokop Z , Damborsky J
Ref : ACS Catal , 9 :4810 , 2019
Abstract : To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Its crystal structure solved to 1.39 A resolution revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen-deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts.
ESTHER : Chaloupkova_2019_ACS.Catal_9_48103
PubMedSearch : Chaloupkova_2019_ACS.Catal_9_48103
PubMedID:
Gene_locus related to this paper: 9zzzz-AncHLDRLuc2 , 9zzzz-AncHLDRLuc , renre-luc

Title : Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase - Jindal_2019_Proc.Natl.Acad.Sci.U.S.A_116_389
Author(s) : Jindal G , Slanska K , Kolev V , Damborsky J , Prokop Z , Warshel A
Ref : Proc Natl Acad Sci U S A , 116 :389 , 2019
Abstract : Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.
ESTHER : Jindal_2019_Proc.Natl.Acad.Sci.U.S.A_116_389
PubMedSearch : Jindal_2019_Proc.Natl.Acad.Sci.U.S.A_116_389
PubMedID: 30587585
Gene_locus related to this paper: xanau-halo1

Title : Crystallization and Crystallographic Analysis of a Bradyrhizobium Elkanii USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site - Pudnikova_2019_Crystals_9_375
Author(s) : Prudnikova T , Kascakova B , Mesters JR , Grinkevich P , Havlickova P , Mazur A , Shaposhnikova A , Chaloupkova R , Damborsky J , Kuty M , Smatanova IK
Ref : , 9 :375 , 2019
Abstract : 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 (DbeADCl) 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. DbeADCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeADCl has been determined and refined to the 1.4 A resolution. The DbeADCl crystals belong to monoclinic space group C121. The DbeADCl molecular structure was characterized and compared with five known haloalkane dehalogenases selected from the Protein Data Bank
ESTHER : Pudnikova_2019_Crystals_9_375
PubMedSearch : Pudnikova_2019_Crystals_9_375
PubMedID:
Gene_locus related to this paper: brael-e2rv62

Title : Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17 - Chrast_2019_Microorganisms_7_
Author(s) : Chrast L , Tratsiak K , Planas-Iglesias J , Daniel L , Prudnikova T , Brezovsky J , Bednar D , Kuta Smatanova I , Chaloupkova R , Damborsky J
Ref : Microorganisms , 7 : , 2019
Abstract : 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 degrees 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.
ESTHER : Chrast_2019_Microorganisms_7_
PubMedSearch : Chrast_2019_Microorganisms_7_
PubMedID: 31661858
Gene_locus related to this paper: 9alte-a3jb27

Title : Crystal structure of the cold-adapted haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5 - Tratsiak_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_324
Author(s) : Tratsiak K , Prudnikova T , Drienovska I , Damborsky J , Brynda J , Pachl P , Kuty M , Chaloupkova R , Rezacova P , Kuta Smatanova I
Ref : Acta Crystallographica F Struct Biol Commun , 75 :324 , 2019
Abstract : 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 A. 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 degrees 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 alpha4 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.
ESTHER : Tratsiak_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_324
PubMedSearch : Tratsiak_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_324
PubMedID: 31045561
Gene_locus related to this paper: psyck-q1qbb9

Title : Computational Study of Protein-Ligand Unbinding for Enzyme Engineering - Marques_2018_Front.Chem_6_650
Author(s) : Marques SM , Bednar D , Damborsky J
Ref : Front Chem , 6 :650 , 2018
Abstract : The computational prediction of unbinding rate constants is presently an emerging topic in drug design. However, the importance of predicting kinetic rates is not restricted to pharmaceutical applications. Many biotechnologically relevant enzymes have their efficiency limited by the binding of the substrates or the release of products. While aiming at improving the ability of our model enzyme haloalkane dehalogenase DhaA to degrade the persistent anthropogenic pollutant 1,2,3-trichloropropane (TCP), the DhaA31 mutant was discovered. This variant had a 32-fold improvement of the catalytic rate toward TCP, but the catalysis became rate-limited by the release of the 2,3-dichloropropan-1-ol (DCP) product from its buried active site. Here we present a computational study to estimate the unbinding rates of the products from DhaA and DhaA31. The metadynamics and adaptive sampling methods were used to predict the relative order of kinetic rates in the different systems, while the absolute values depended significantly on the conditions used (method, force field, and water model). Free energy calculations provided the energetic landscape of the unbinding process. A detailed analysis of the structural and energetic bottlenecks allowed the identification of the residues playing a key role during the release of DCP from DhaA31 via the main access tunnel. Some of these hot-spots could also be identified by the fast CaverDock tool for predicting the transport of ligands through tunnels. Targeting those hot-spots by mutagenesis should improve the unbinding rates of the DCP product and the overall catalytic efficiency with TCP.
ESTHER : Marques_2018_Front.Chem_6_650
PubMedSearch : Marques_2018_Front.Chem_6_650
PubMedID: 30671430

Title : Detection of Chloroalkanes by Surface-Enhanced Raman Spectroscopy in Microfluidic Chips - Pilat_2018_Sensors.(Basel)_18_
Author(s) : Pilat Z , Kizovsky M , Jezek J , Kratky S , Sobota J , Siler M , Samek O , Buryska T , Vanacek P , Damborsky J , Prokop Z , Zemanek P
Ref : Sensors (Basel) , 18 : , 2018
Abstract : Optofluidics, a research discipline combining optics with microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g., for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.
ESTHER : Pilat_2018_Sensors.(Basel)_18_
PubMedSearch : Pilat_2018_Sensors.(Basel)_18_
PubMedID: 30249041

Title : Sensitive operation of enzyme-based biodevices by advanced signal processing - Mazurenko_2018_PLoS.One_13_e0198913
Author(s) : Mazurenko S , Bidmanova S , Kotlanova M , Damborsky J , Prokop Z
Ref : PLoS ONE , 13 :e0198913 , 2018
Abstract : Analytical devices that combine sensitive biological component with a physicochemical detector hold a great potential for various applications, e.g., environmental monitoring, food analysis or medical diagnostics. Continuous efforts to develop inexpensive sensitive biodevices for detecting target substances typically focus on the design of biorecognition elements and their physical implementation, while the methods for processing signals generated by such devices have received far less attention. Here, we present fundamental considerations related to signal processing in biosensor design and investigate how undemanding signal treatment facilitates calibration and operation of enzyme-based biodevices. Our signal treatment approach was thoroughly validated with two model systems: (i) a biodevice for detecting chemical warfare agents and environmental pollutants based on the activity of haloalkane dehalogenase, with the sensitive range for bis(2-chloroethyl) ether of 0.01-0.8 mM and (ii) a biodevice for detecting hazardous pesticides based on the activity of gamma-hexachlorocyclohexane dehydrochlorinase with the sensitive range for gamma-hexachlorocyclohexane of 0.01-0.3 mM. We demonstrate that the advanced signal processing based on curve fitting enables precise quantification of parameters important for sensitive operation of enzyme-based biodevices, including: (i) automated exclusion of signal regions with substantial noise, (ii) derivation of calibration curves with significantly reduced error, (iii) shortening of the detection time, and (iv) reliable extrapolation of the signal to the initial conditions. The presented simple signal curve fitting supports rational design of optimal system setup by explicit and flexible quantification of its properties and will find a broad use in the development of sensitive and robust biodevices.
ESTHER : Mazurenko_2018_PLoS.One_13_e0198913
PubMedSearch : Mazurenko_2018_PLoS.One_13_e0198913
PubMedID: 29912920

Title : Development of fluorescent assay for monitoring of dehalogenase activity - Nevolova_2018_Biotechnol.J__e1800144
Author(s) : Nevolova S , Manaskova E , Mazurenko S , Damborsky J , Prokop Z
Ref : Biotechnol J , :e1800144 , 2018
Abstract : The rapid accumulation of sequence data and powerful protein engineering techniques providing a large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Here we report a continuous assay for screening of enzymatic activity. The assay was developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of derivative of 8-hydroxypyrene-1,3,6-trisulphonic acid due to the pH drop associated with the dehalogenation reactions. The assay is performed in a microplate format using a purified enzyme, cell-free extract or intact cells, making the analysis quick and simple. The method exhibits high sensitivity with a limit of detection of 0.06 mM. The assay has been successfully validated with gas chromatography and then applied for screening of twelve haloalkane dehalogenases with the environmental pollutant bis(2-chloroethyl) ether and chemical warfare agent sulfur mustard. Six enzymes exhibited detectable activity with both substrates. The within-day variability of the assay for five replicates (n = 5) was 21%.
ESTHER : Nevolova_2018_Biotechnol.J__e1800144
PubMedSearch : Nevolova_2018_Biotechnol.J__e1800144
PubMedID: 30052322

Title : Conformational changes allow processing of bulky substrates by a haloalkane dehalogenase with a small and buried active site - Kokkonen_2018_J.Biol.Chem_293_11505
Author(s) : Kokkonen P , Bednar D , Dockalova V , Prokop Z , Damborsky J
Ref : Journal of Biological Chemistry , 293 :11505 , 2018
Abstract : Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 A(3)) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 A(3)). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.
ESTHER : Kokkonen_2018_J.Biol.Chem_293_11505
PubMedSearch : Kokkonen_2018_J.Biol.Chem_293_11505
PubMedID: 29858243
Gene_locus related to this paper: xanau-halo1

Title : A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity - Buryska_2018_Appl.Environ.Microbiol_84_
Author(s) : Buryska T , Babkova P , Vavra O , Damborsky J , Prokop Z
Ref : Applied Environmental Microbiology , 84 : , 2018
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.
ESTHER : Buryska_2018_Appl.Environ.Microbiol_84_
PubMedSearch : Buryska_2018_Appl.Environ.Microbiol_84_
PubMedID: 29101190
Gene_locus related to this paper: 9cyan-q6dnd9

Title : Impact of the access tunnel engineering on catalysis is strictly ligand-specific - Kaushik_2018_FEBS.J_285_1456
Author(s) : Kaushik S , Marques SM , Khirsariya P , Paruch K , Libichova L , Brezovsky J , Prokop Z , Chaloupkova R , Damborsky J
Ref : Febs J , 285 :1456 , 2018
Abstract : The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.
ESTHER : Kaushik_2018_FEBS.J_285_1456
PubMedSearch : Kaushik_2018_FEBS.J_285_1456
PubMedID: 29478278

Title : Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear beta-Haloalkanes: Open-Solvated versus Occluded-Desolvated Active Sites - Liskova_2017_Angew.Chem.Int.Ed.Engl_56_4719
Author(s) : Liskova V , Stepankova V , Bednar D , Brezovsky J , Prokop Z , Chaloupkova R , Damborsky J
Ref : Angew Chem Int Ed Engl , 56 :4719 , 2017
Abstract : The enzymatic enantiodiscrimination of linear beta-haloalkanes is difficult because the simple structures of the substrates prevent directional interactions. Herein we describe two distinct molecular mechanisms for the enantiodiscrimination of the beta-haloalkane 2-bromopentane by haloalkane dehalogenases. Highly enantioselective DbjA has an open, solvent-accessible active site, whereas the engineered enzyme DhaA31 has an occluded and less solvated cavity but shows similar enantioselectivity. The enantioselectivity of DhaA31 arises from steric hindrance imposed by two specific substitutions rather than hydration as in DbjA.
ESTHER : Liskova_2017_Angew.Chem.Int.Ed.Engl_56_4719
PubMedSearch : Liskova_2017_Angew.Chem.Int.Ed.Engl_56_4719
PubMedID: 28334478

Title : Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity - Babkova_2017_Chembiochem_18_1448
Author(s) : Babkova P , Sebestova E , Brezovsky J , Chaloupkova R , Damborsky J
Ref : Chembiochem , 18 :1448 , 2017
Abstract : Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (DeltaTm up to 24 degrees C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.
ESTHER : Babkova_2017_Chembiochem_18_1448
PubMedSearch : Babkova_2017_Chembiochem_18_1448
PubMedID: 28419658
Gene_locus related to this paper: 9bact-AncHLD5

Title : Metagenome-derived haloalkane dehalogenases with novel catalytic properties - Kotik_2017_Appl.Microbiol.Biotechnol_101_6385
Author(s) : Kotik M , Vanacek P , Kunka A , Prokop Z , Damborsky J
Ref : Applied Microbiology & Biotechnology , 101 :6385 , 2017
Abstract : Haloalkane dehalogenases (HLDs) are environmentally relevant enzymes cleaving a carbon-halogen bond in a wide range of halogenated pollutants. PCR with degenerate primers and genome-walking was used for the retrieval of four HLD-encoding genes from groundwater-derived environmental DNA. Using specific primers and the environmental DNA as a template, we succeeded in generating additional amplicons, resulting altogether in three clusters of sequences with each cluster comprising 8-13 closely related putative HLD-encoding genes. A phylogenetic analysis of the translated genes revealed that three HLDs are members of the HLD-I subfamily, whereas one gene encodes an enzyme from the subfamily HLD-II. Two metagenome-derived HLDs, eHLD-B and eHLD-C, each from a different subfamily, were heterologously produced in active form, purified and characterized in terms of their thermostability, pH and temperature optimum, quaternary structure, substrate specificity towards 30 halogenated compounds, and enantioselectivity. eHLD-B and eHLD-C showed striking differences in their activities, substrate preferences, and tolerance to temperature. Profound differences were also determined in the enantiopreference and enantioselectivity of these enzymes towards selected substrates. Comparing our data with those of known HLDs revealed that eHLD-C exhibits a unique combination of high thermostability, high activity, and an unusually broad pH optimum, which covers the entire range of pH 5.5-8.9. Moreover, a so far unreported high thermostability for HLDs was determined for this enzyme at pH values lower than 6.0. Thus, eHLD-C represents an attractive and novel biocatalyst for biotechnological applications.
ESTHER : Kotik_2017_Appl.Microbiol.Biotechnol_101_6385
PubMedSearch : Kotik_2017_Appl.Microbiol.Biotechnol_101_6385
PubMedID: 28674849
Gene_locus related to this paper: 9bact-v5ln96 , 9bact-v5llz5 , 9bact-v5llk8

Title : Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling - Marques_2017_J.Chem.Inf.Model_57_1970
Author(s) : Marques SM , Dunajova Z , Prokop Z , Chaloupkova R , Brezovsky J , Damborsky J
Ref : J Chem Inf Model , 57 :1970 , 2017
Abstract : The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the SN2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust strategies in the development of new biocatalysts.
ESTHER : Marques_2017_J.Chem.Inf.Model_57_1970
PubMedSearch : Marques_2017_J.Chem.Inf.Model_57_1970
PubMedID: 28696117

Title : Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels - Kaushik_2017_FEBS.J_284_134
Author(s) : Kaushik S , Prokop Z , Damborsky J , Chaloupkova R
Ref : Febs J , 284 :134 , 2017
Abstract : Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as alpha/beta-hydrolases containing > 100 000 protein sequences based on the Pfam database.
ESTHER : Kaushik_2017_FEBS.J_284_134
PubMedSearch : Kaushik_2017_FEBS.J_284_134
PubMedID: 27863020

Title : Engineering a de novo transport tunnel - Brezovsky_2016_ACS.Catal_6_7597
Author(s) : Brezovsky J , Babkova P , Degtjarik O , Fortova A , Gora A , Iermak I , Rezacova P , Dvorak P , Kuta-Smatanova I , Prokop Z , Chaloupkova R , Damborsky J
Ref : ACS Catal , 6 :7597 , 2016
Abstract : Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional -Yhole into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members, up to 32-times increased substrate inhibition and 17-times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to the native enzyme, and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s-1 with 1,2-dibromoethane at 37oC and pH=8.6). Crystallographic analysis and molecular dynamics simulations confirmed successful introduction of structur-ally defined and functional transport tunnel. Our study demonstrates that whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels and we anticipate that this engineering strategy will facilitate creation of a wide range of useful biocatalysts.
ESTHER : Brezovsky_2016_ACS.Catal_6_7597
PubMedSearch : Brezovsky_2016_ACS.Catal_6_7597
PubMedID:
Gene_locus related to this paper: sphpi-linb

Title : Fluorescence-based biosensor for monitoring of environmental pollutants: From concept to field application - Bidmanova_2016_Biosens.Bioelectron_84_97
Author(s) : Bidmanova S , Kotlanova M , Rataj T , Damborsky J , Trtilek M , Prokop Z
Ref : Biosensors & Bioelectronics , 84 :97 , 2016
Abstract : An advanced optical biosensor was developed based on the enzymatic reaction with halogenated aliphatic hydrocarbons that is accompanied by the fluorescence change of pH indicator. The device is applicable for the detection of halogenated contaminants in water samples with pH ranging from 4 to 10 and temperature ranging from 5 to 60 degrees C. Main advantages of the developed biosensor are small size (60x30x190mm(3)) and portability, which together with short measurement time of 1min belong to crucial attributes of analytical technique useful for routine environmental monitoring. The biosensor was successfully applied for the detection of several important halogenated pollutants under laboratory conditions, e.g., 1,2-dichloroethane, 1,2,3-trichloropropane and gamma-hexachlorocyclohexane, with the limits of detection of 2.7, 1.4 and 12.1mgL(-1), respectively. The continuous monitoring was demonstrated by repetitive injection of halogenated compound into measurement solution. Consequently, field trials under environmental settings were performed. The presence of 1,2-dichloroethane (10mgL(-1)) was proved unambiguously on one of three potentially contaminated sites in Czech Republic, and the same contaminant was monitored on contaminated locality in Serbia. Equipped by Global Positioning System, the biosensor was used for creation of a precise map of contamination. Concentrations determined by biosensor and by gas chromatograph coupled with mass spectrometer exhibited the correlation coefficient of 0.92, providing a good confidence for the routine use of the biosensor system in both field screening and monitoring.
ESTHER : Bidmanova_2016_Biosens.Bioelectron_84_97
PubMedSearch : Bidmanova_2016_Biosens.Bioelectron_84_97
PubMedID: 26725215

Title : Suppression of protein inactivation during freezing by minimizing pH changes using ionic cryoprotectants - Krauskova_2016_Int.J.Pharm_509_41
Author(s) : Krauskova L , Prochazkova J , Klaskova M , Filipova L , Chaloupkova R , Maly S , Damborsky J , Heger D
Ref : Int J Pharm , 509 :41 , 2016
Abstract : Freezing and lyophilization are often used for stabilization of biomolecules; however, this sometimes results in partial degradation and loss of biological function in these molecules. In this study we examined the effect of freezing-induced acidity changes on denaturation of the model enzyme haloalkane dehalogenase under various experimental conditions. The effective local pH of frozen solutions is shown to be the key causal factor in protein stability. To preserve the activity of frozen-thawed enzymes, acidity changes were prevented by the addition of an ionic cryoprotectant, a compound which counteracts pH changes during freezing due to selective incorporation of its ions into the ice. This approach resulted in complete recovery of enzyme activity after multiple freeze-thaw cycles. We propose the utilization of ionic cryoprotectants as a new and effective cryopreservation method in research laboratories as well as in industrial processes.
ESTHER : Krauskova_2016_Int.J.Pharm_509_41
PubMedSearch : Krauskova_2016_Int.J.Pharm_509_41
PubMedID: 27224008

Title : Discovery of Novel Haloalkane Dehalogenase Inhibitors - Buryska_2016_Appl.Environ.Microbiol_82_1958
Author(s) : Buryska T , Daniel L , Kunka A , Brezovsky J , Damborsky J , Prokop Z
Ref : Applied Environmental Microbiology , 82 :1958 , 2016
Abstract : Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 muM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization.
ESTHER : Buryska_2016_Appl.Environ.Microbiol_82_1958
PubMedSearch : Buryska_2016_Appl.Environ.Microbiol_82_1958
PubMedID: 26773086

Title : FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants - Bednar_2015_PLoS.Comput.Biol_11_e1004556
Author(s) : Bednar D , Beerens K , Sebestova E , Bendl J , Khare S , Chaloupkova R , Prokop Z , Brezovsky J , Baker D , Damborsky J
Ref : PLoS Comput Biol , 11 :e1004556 , 2015
Abstract : There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and gamma-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (DeltaTm = 24 degrees C and 21 degrees C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.
ESTHER : Bednar_2015_PLoS.Comput.Biol_11_e1004556
PubMedSearch : Bednar_2015_PLoS.Comput.Biol_11_e1004556
PubMedID: 26529612
Gene_locus related to this paper: rhoso-halo1

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
PubMedID:
Gene_locus related to this paper: rhoso-halo1

Title : Mechanism-based discovery of novel substrates of haloalkane dehalogenases using in silico screening - Daniel_2015_J.Chem.Inf.Model_55_54
Author(s) : Daniel L , Buryska T , Prokop Z , Damborsky J , Brezovsky J
Ref : J Chem Inf Model , 55 :54 , 2015
Abstract : 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 compounds-true substrates and binders-for 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.
ESTHER : Daniel_2015_J.Chem.Inf.Model_55_54
PubMedSearch : Daniel_2015_J.Chem.Inf.Model_55_54
PubMedID: 25495415

Title : Site-specific analysis of protein hydration based on unnatural amino acid fluorescence - Amaro_2015_J.Am.Chem.Soc_137_4988
Author(s) : Amaro M , Brezovsky J , Kovacova S , Sykora J , Bednar D , Nemec V , Liskova V , Kurumbang NP , Beerens K , Chaloupkova R , Paruch K , Hof M , Damborsky J
Ref : Journal of the American Chemical Society , 137 :4988 , 2015
Abstract : Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.
ESTHER : Amaro_2015_J.Am.Chem.Soc_137_4988
PubMedSearch : Amaro_2015_J.Am.Chem.Soc_137_4988
PubMedID: 25815779

Title : Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31 - Lahoda_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_209
Author(s) : Lahoda M , Mesters JR , Stsiapanava A , Chaloupkova R , Kuty M , Damborsky J , Kuta Smatanova I
Ref : Acta Crystallographica D Biol Crystallogr , 70 :209 , 2014
Abstract : Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 A resolution crystal structure of substrate-free DhaA31, the 1.26 A resolution structure of DhaA31 in complex with TCP and the 1.95 A resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.
ESTHER : Lahoda_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_209
PubMedSearch : Lahoda_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_209
PubMedID: 24531456
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 : Microscopic monitoring provides information on structure and properties during biocatalyst immobilization - Bidmanova_2014_Biotechnol.J_9_852
Author(s) : Bidmanova S , Hrdlickova E , Jaros J , Ilkovics L , Hampl A , Damborsky J , Prokop Z
Ref : Biotechnol J , 9 :852 , 2014
Abstract : Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques - bright-field, confocal and electron microscopy - were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non-treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions.
ESTHER : Bidmanova_2014_Biotechnol.J_9_852
PubMedSearch : Bidmanova_2014_Biotechnol.J_9_852
PubMedID: 24639415

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 : A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation - Samin_2014_Appl.Environ.Microbiol_80_5467
Author(s) : Samin G , Pavlova M , Arif MI , Postema CP , Damborsky J , Janssen DB
Ref : Applied Environmental Microbiology , 80 :5467 , 2014
Abstract : 1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.
ESTHER : Samin_2014_Appl.Environ.Microbiol_80_5467
PubMedSearch : Samin_2014_Appl.Environ.Microbiol_80_5467
PubMedID: 24973068

Title : Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards beta-hexachlorocyclohexane - Moriuchi_2014_AMB.Express_4_72
Author(s) : Moriuchi R , Tanaka H , Nikawadori Y , Ishitsuka M , Ito M , Ohtsubo Y , Tsuda M , Damborsky J , Prokop Z , Nagata Y
Ref : AMB Express , 4 :72 , 2014
Abstract : Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards beta-hexachlorocyclohexane (beta-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's beta-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.
ESTHER : Moriuchi_2014_AMB.Express_4_72
PubMedSearch : Moriuchi_2014_AMB.Express_4_72
PubMedID: 25401073

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 : Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents - Stepankova_2013_Chembiochem_14_890
Author(s) : Stepankova V , Khabiri M , Brezovsky J , Pavelka A , Sykora J , Amaro M , Minofar B , Prokop Z , Hof M , Ettrich R , Chaloupkova R , Damborsky J
Ref : Chembiochem , 14 :890 , 2013
Abstract : The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration.
ESTHER : Stepankova_2013_Chembiochem_14_890
PubMedSearch : Stepankova_2013_Chembiochem_14_890
PubMedID: 23564727
Gene_locus related to this paper: rhoso-halo1

Title : Differences in crystallization of two LinB variants from Sphingobium japonicum UT26 - Degtjarik_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_284
Author(s) : Degtjarik O , Chaloupkova R , Rezacova P , Kuty M , Damborsky J , Kuta Smatanova I
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 69 :284 , 2013
Abstract : Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 A. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.
ESTHER : Degtjarik_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_284
PubMedSearch : Degtjarik_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_284
PubMedID: 23519805
Gene_locus related to this paper: sphpi-linb

Title : Interaction of organic solvents with protein structures at protein-solvent interface - Khabiri_2013_J.Mol.Model_19_4701
Author(s) : Khabiri M , Minofar B , Brezovsky J , Damborsky J , Ettrich R
Ref : J Mol Model , 19 :4701 , 2013
Abstract : The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.
ESTHER : Khabiri_2013_J.Mol.Model_19_4701
PubMedSearch : Khabiri_2013_J.Mol.Model_19_4701
PubMedID: 22760789

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 3.8.1.5, 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 : Release of halide ions from the buried active site of the haloalkane dehalogenase LinB revealed by stopped-flow fluorescence analysis and free energy calculations - Hladilkova_2013_J.Phys.Chem.B_117_14329
Author(s) : Hladilkova J , Prokop Z , Chaloupkova R , Damborsky J , Jungwirth P
Ref : J Phys Chem B , 117 :14329 , 2013
Abstract : 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. Our study contributes toward understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies.
ESTHER : Hladilkova_2013_J.Phys.Chem.B_117_14329
PubMedSearch : Hladilkova_2013_J.Phys.Chem.B_117_14329
PubMedID: 24151979

Title : The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 - Hasan_2013_FEBS.J_280_3149
Author(s) : Hasan K , Gora A , Brezovsky J , Chaloupkova R , Moskalikova H , Fortova A , Nagata Y , Damborsky J , Prokop Z
Ref : Febs J , 280 :3149 , 2013
Abstract : 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 K(0.5) 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.
ESTHER : Hasan_2013_FEBS.J_280_3149
PubMedSearch : Hasan_2013_FEBS.J_280_3149
PubMedID: 23490078

Title : Are time-dependent fluorescence shifts at the tunnel mouth of haloalkane dehalogenase enzymes dependent on the choice of the chromophore? - Amaro_2013_J.Phys.Chem.B_117_7898
Author(s) : Amaro M , Brezovsky J , Kovacova S , Maier L , Chaloupkova R , Sykora J , Paruch K , Damborsky J , Hof M
Ref : J Phys Chem B , 117 :7898 , 2013
Abstract : Time-dependent fluorescence shifts (TDFS) of chromophores selectively attached to proteins may give information on the dynamics of the probed protein moieties and their degree of hydration. Previously, we demonstrated that a coumarin dye selectively labeling the tunnel mouth of different haloalkane dehalogenases (HLDs) can distinguish between different widths of tunnel mouth openings. In order to generalize those findings analogous experiments were performed using a different chromophore probing the same region of these enzymes. To this end we synthesized and characterized three new fluorescent probes derived from dimethylaminonaphthalene bearing a linker almost identical to that of the coumarin dye used in our previous study. Labeling efficiencies, acrylamide quenching, fluorescence anisotropies, and TDFS for the examined fluorescent substrates confirm the picture gained from the coumarin studies: the different tunnel mouth opening, predicted by crystal structures, is reflected in the hydration and tunnel mouth dynamics of the investigated HLDs. Comparison of the TDFS reported by the coumarin dye with those obtained with the new dimethylaminonaphthalene dyes shows that the choice of chromophore may strongly influence the recorded TDFS characteristics. The intrinsic design of our labeling strategy and the variation of the linker length ensure that both dyes probe the identical enzyme region; moreover, the covalently fixed position of the chromophore does not allow for a major relocalization within the HLD structures. Our study shows, for the first time, that TDFS may strongly depend on the choice of the chromophore, even though the identical region of a protein is explored.
ESTHER : Amaro_2013_J.Phys.Chem.B_117_7898
PubMedSearch : Amaro_2013_J.Phys.Chem.B_117_7898
PubMedID: 23781851

Title : Crystallographic analysis of new psychrophilic haloalkane dehalogenases: DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17 - Tratsiak_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_683
Author(s) : Tratsiak K , Degtjarik O , Drienovska I , Chrast L , Rezacova P , Kuty M , Chaloupkova R , Damborsky J , Kuta Smatanova I
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 69 :683 , 2013
Abstract : Haloalkane dehalogenases are hydrolytic enzymes with a broad range of potential practical applications such as biodegradation, biosensing, biocatalysis and cellular imaging. Two newly isolated psychrophilic haloalkane dehalogenases exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were purified and used for crystallization experiments. After the optimization of crystallization conditions, crystals of diffraction quality were obtained. Diffraction data sets were collected for native enzymes and complexes with selected ligands such as 1-bromohexane and 1,2-dichloroethane to resolutions ranging from 1.05 to 2.49 A.
ESTHER : Tratsiak_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_683
PubMedSearch : Tratsiak_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_683
PubMedID: 23722854
Gene_locus related to this paper: 9alte-a3jb27 , psyck-q1qbb9

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
PubMedID:

Title : Cation-specific effects on enzymatic catalysis driven by interactions at the tunnel mouth - Stepankova_2013_J.Phys.Chem.B_117_6394
Author(s) : Stepankova V , Paterova J , Damborsky J , Jungwirth P , Chaloupkova R , Heyda J
Ref : J Phys Chem B , 117 :6394 , 2013
Abstract : Cationic specificity which follows the Hofmeister series has been established for the catalytic efficiency of haloalkane dehalogenase LinB by a combination of molecular dynamics simulations and enzyme kinetic experiments. Simulations provided a detailed molecular picture of cation interactions with negatively charged residues on the protein surface, particularly at the tunnel mouth leading to the enzyme active site. On the basis of the binding affinities, cations were ordered as Na(+) > K(+) > Rb(+) > Cs(+). In agreement with this result, a steady-state kinetic analysis disclosed that the smaller alkali cations influence formation and productivity of enzyme-substrate complexes more efficiently than the larger ones. A subsequent systematic investigation of two LinB mutants with engineered charge in the cation-binding site revealed that the observed cation affinities are enhanced by increasing the number of negatively charged residues at the tunnel mouth, and vice versa, reduced by decreasing this number. However, the cation-specific effects are overwhelmed by strong electrostatic interactions in the former case. Interestingly, the substrate inhibition of the mutant LinB L177D in the presence of chloride salts was 7 times lower than that of LinB wild type in glycine buffer. Our work provides new insight into the mechanisms of specific cation effects on enzyme activity and suggests a potential strategy for suppression of substrate inhibition by the combination of protein and medium engineering.
ESTHER : Stepankova_2013_J.Phys.Chem.B_117_6394
PubMedSearch : Stepankova_2013_J.Phys.Chem.B_117_6394
PubMedID: 23627286

Title : A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB - Biedermannova_2012_J.Biol.Chem_287_29062
Author(s) : Biedermannova L , Prokop Z , Gora A , Chovancova E , Kovacs M , Damborsky J , Wade RC
Ref : Journal of Biological Chemistry , 287 :29062 , 2012
Abstract : Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.
ESTHER : Biedermannova_2012_J.Biol.Chem_287_29062
PubMedSearch : Biedermannova_2012_J.Biol.Chem_287_29062
PubMedID: 22745119

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 : Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane - Lahoda_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_397
Author(s) : Lahoda M , Chaloupkova R , Stsiapanava A , Damborsky J , Kuta Smatanova I
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 67 :397 , 2011
Abstract : Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 A for DhaA31 and to 1.26 A for DhaA31 complexed with TCP.
ESTHER : Lahoda_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_397
PubMedSearch : Lahoda_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_397
PubMedID: 21393851
Gene_locus related to this paper: rhoso-halo1

Title : Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands - Stsiapanava_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_253
Author(s) : Stsiapanava A , Chaloupkova R , Fortova A , Brynda J , Weiss MS , Damborsky J , Smatanova IK
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 67 :253 , 2011
Abstract : Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 A resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 A resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 A, respectively.
ESTHER : Stsiapanava_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_253
PubMedSearch : Stsiapanava_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_253
PubMedID: 21301099
Gene_locus related to this paper: rhoso-halo1

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 : Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature - Chaloupkova_2011_FEBS.J_278_2728
Author(s) : Chaloupkova R , Prokop Z , Sato Y , Nagata Y , Damborsky J
Ref : Febs J , 278 :2728 , 2011
Abstract : The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 +/- 0.2 degrees C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 degrees C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Delta(R-S)DeltaH and Delta(R-S)DeltaS were determined. The thermodynamic analysis revealed that the resolution of beta-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of alpha-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.
ESTHER : Chaloupkova_2011_FEBS.J_278_2728
PubMedSearch : Chaloupkova_2011_FEBS.J_278_2728
PubMedID: 21635695

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 : Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons - Bidmanova_2010_Anal.Bioanal.Chem_398_1891
Author(s) : Bidmanova S , Chaloupkova R , Damborsky J , Prokop Z
Ref : Anal Bioanal Chem , 398 :1891 , 2010
Abstract : An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants.
ESTHER : Bidmanova_2010_Anal.Bioanal.Chem_398_1891
PubMedSearch : Bidmanova_2010_Anal.Bioanal.Chem_398_1891
PubMedID: 20721539

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 : Editorial: Protein design and engineering for biocatalysis -
Author(s) : Damborsky J
Ref : Biotechnol J , 4 :439 , 2009
PubMedID: 19370730

Title : Computational tools for designing and engineering biocatalysts - Damborsky_2009_Curr.Opin.Chem.Biol_13_26
Author(s) : Damborsky J , Brezovsky J
Ref : Curr Opin Chemical Biology , 13 :26 , 2009
Abstract : Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are reviewed. The applications of these tools for de novo design of protein active sites, optimization of substrate access and product exit pathways, redesign of protein-protein interfaces, identification of neutral/advantageous/deleterious mutations in the libraries from directed evolution and stabilization of protein structures are described. Remarkable progress is seen in de novo design of enzymes catalyzing a chemical reaction for which a natural biocatalyst does not exist. Yet, constructed biocatalysts do not match natural enzymes in their efficiency, suggesting that more research is needed to capture all the important features of natural biocatalysts in theoretical designs.
ESTHER : Damborsky_2009_Curr.Opin.Chem.Biol_13_26
PubMedSearch : Damborsky_2009_Curr.Opin.Chem.Biol_13_26
PubMedID: 19297237

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 : Nanosecond time-dependent Stokes shift at the tunnel mouth of haloalkane dehalogenases - Jesenska_2009_J.Am.Chem.Soc_131_494
Author(s) : Jesenska A , Sykora J , Olzynska A , Brezovsky J , Zdrahal Z , Damborsky J , Hof M
Ref : Journal of the American Chemical Society , 131 :494 , 2009
Abstract : 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 obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.
ESTHER : Jesenska_2009_J.Am.Chem.Soc_131_494
PubMedSearch : Jesenska_2009_J.Am.Chem.Soc_131_494
PubMedID: 19113888

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 : Crystallization and preliminary X-ray analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94 - Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
Author(s) : Prudnikova T , Mozga T , Rezacova P , Chaloupkova R , Sato Y , Nagata Y , Brynda J , Kuty M , Damborsky J , Smatanova IK
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 65 :353 , 2009
Abstract : 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 P2(1)2(1)2(1), 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.
ESTHER : Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
PubMedSearch : Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
PubMedID: 19342778
Gene_locus related to this paper: brael-e2rv62

Title : Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate - Pavlova_2009_Nat.Chem.Biol_5_727
Author(s) : Pavlova M , Klvana M , Prokop Z , Chaloupkova R , Banas P , Otyepka M , Wade RC , Tsuda M , Nagata Y , Damborsky J
Ref : Nat Chemical Biology , 5 :727 , 2009
Abstract : Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
ESTHER : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedSearch : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedID: 19701186

Title : Second step of hydrolytic dehalogenation in haloalkane dehalogenase investigated by QM\/MM methods - Otyepka_2008_Proteins_70_707
Author(s) : Otyepka M , Banas P , Magistrato A , Carloni P , Damborsky J
Ref : Proteins , 70 :707 , 2008
Abstract : Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5 +/- 2 kcal . mol(-1). The lowering of the energy barrier of catalyzed reaction with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.
ESTHER : Otyepka_2008_Proteins_70_707
PubMedSearch : Otyepka_2008_Proteins_70_707
PubMedID: 17729274

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

Title : Stepwise dissection and visualization of the catalytic mechanism of haloalkane dehalogenase LinB using molecular dynamics simulations and computer graphics - Negri_2007_J.Mol.Graph.Model_26_643
Author(s) : Negri A , Marco E , Damborsky J , Gago F
Ref : J Mol Graph Model , 26 :643 , 2007
Abstract : The different steps of the dehalogenation reaction carried out by LinB on three different substrates have been characterized using a combination of quantum mechanical calculations and molecular dynamics simulations. This has allowed us to obtain information in atomic detail about each step of the reaction mechanism, that is, substrate entrance and achievement of the near-attack conformation, transition state stabilization within the active site, halide stabilization, water molecule activation and subsequent hydrolytic attack on the ester intermediate with formation of alcohol, and finally product release. Importantly, no bias or external forces were applied during the whole procedure so that both intermediates and products were completely free to sample configuration space in order to adapt to the plasticity of the active site and/or search for an exit. Differences in substrate reactivity were found to be correlated with the ease of adopting the near-attack conformation and two different exit pathways were found for product release that do not interfere with substrate entrance. Additional support for the different entry and exit pathways was independently obtained from an examination of the enzyme's normal modes.
ESTHER : Negri_2007_J.Mol.Graph.Model_26_643
PubMedSearch : Negri_2007_J.Mol.Graph.Model_26_643
PubMedID: 17451982

Title : The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution - Pavlova_2007_J.Struct.Biol_157_384
Author(s) : Pavlova M , Klvana M , Jesenska A , Prokop Z , Konecna H , Sato T , Tsuda M , Nagata Y , Damborsky J
Ref : J Struct Biol , 157 :384 , 2007
Abstract : Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.
ESTHER : Pavlova_2007_J.Struct.Biol_157_384
PubMedSearch : Pavlova_2007_J.Struct.Biol_157_384
PubMedID: 17084094

Title : Degradation of beta-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205 - Ito_2007_Arch.Microbiol_188_313
Author(s) : Ito M , Prokop Z , Klvana M , Otsubo Y , Tsuda M , Damborsky J , Nagata Y
Ref : Arch Microbiol , 188 :313 , 2007
Abstract : The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal gamma-isomer and noninsecticidal alpha-, beta-, and delta-isomers, among which beta-HCH is the most recalcitrant and has caused serious environmental problems. A gamma-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded beta-HCH more rapidly than the well-characterized gamma-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, beta-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB(MI)) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB(UT)) was identified as an enzyme responsible for the two-step conversion of beta-HCH to TCDL. This property of LinB(MI) contrasted with that of LinB(UT), which catalyzed only the first step conversion of beta-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB(MI). However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB(MI). Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB (MI) gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.
ESTHER : Ito_2007_Arch.Microbiol_188_313
PubMedSearch : Ito_2007_Arch.Microbiol_188_313
PubMedID: 17516046
Gene_locus related to this paper: sphpi-linb

Title : Weak activity of haloalkane dehalogenase LinB with 1,2,3-trichloropropane revealed by X-Ray crystallography and microcalorimetry - Monincova_2007_Appl.Environ.Microbiol_73_2005
Author(s) : Monincova M , Prokop Z , Vevodova J , Nagata Y , Damborsky J
Ref : Applied Environmental Microbiology , 73 :2005 , 2007
Abstract : 1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.
ESTHER : Monincova_2007_Appl.Environ.Microbiol_73_2005
PubMedSearch : Monincova_2007_Appl.Environ.Microbiol_73_2005
PubMedID: 17259360
Gene_locus related to this paper: sphpi-linb

Title : Exploring the binding sites of the haloalkane dehalogenase DhlA from Xanthobacter autotrophicus GJ10 - Silberstein_2007_Biochemistry_46_9239
Author(s) : Silberstein M , Damborsky J , Vajda S
Ref : Biochemistry , 46 :9239 , 2007
Abstract : The catalytic site of haloalkane dehalogenase DhlA is buried more than 10 A from the protein surface. While potential access channels to this site have been reported, the precise mechanism of substrate import and product export is still unconfirmed. We used computational methods to examine surface pockets and their putative roles in ligand access to and from the catalytic site. Computational solvent mapping moves small organic molecule as probes over the protein surface in order to identify energetically favorable sites, that is, regions that tend to bind a variety of molecules. The mapping of three DhlA structures identifies seven such regions, some of which have been previously suggested to be involved in the binding and the import/export of substrates or products. These sites are the active site, the putative entrance of the channel leading to the active site, two pockets that bind Br- ions, a pocket in the slot region, and two additional sites between the main domain and the cap of DhlA. We also performed mapping and free energy analysis of the DhlA structures using the substrate, 1,2-dichloroethane, and halide ions as probes. The findings were compared to crystallographic data and to results obtained by CAVER, a program developed for finding routes from protein clefts and cavities to the surface. Solvent mapping precisely reproduced all three Br- binding sites identified by protein crystallography and the openings to four channels found by CAVER. The analyses suggest that (i) the active site has the highest affinity for the substrate molecule, (ii) the substrate initially binds at the entrance of the main tunnel, (iii) the site Br2, close to the entrance, is likely to serve as an intermediate binding site in product export, (iv) the site Br3, induced in the structure at high concentrations of Br-, could be part of an auxiliary route for product release, and (v) three of the identified sites are likely to be entrances of water-access channels leading to the active site. For comparison, we also mapped haloalkane dehalogenases DhaA and LinB, both of which contain significantly larger and more solvent accessible binding sites than DhlA. The mapping of DhaA and LinB places the majority of probes in the active site, but most of the other six regions consistently identified in DhlA were not observed, suggesting that the more open active site eliminates the need for intermediate binding sites for the collision complex seen in DhlA.
ESTHER : Silberstein_2007_Biochemistry_46_9239
PubMedSearch : Silberstein_2007_Biochemistry_46_9239
PubMedID: 17645312
Gene_locus related to this paper: xanau-halo1

Title : Crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110 - Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
Author(s) : Sato Y , Natsume R , Tsuda M , Damborsky J , Nagata Y , Senda T
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :294 , 2007
Abstract : 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 beta-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 P2(1)2(1)2, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 A. The crystal diffracts to 1.75 A resolution.
ESTHER : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedSearch : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedID: 17401198

Title : Phylogenetic analysis of haloalkane dehalogenases - Chovancova_2007_Proteins_67_305
Author(s) : Chovancova E , Kosinski J , Bujnicki JM , Damborsky J
Ref : Proteins , 67 :305 , 2007
Abstract : Haloalkane dehalogenases (HLDs) are enzymes that catalyze the cleavage of carbon-halogen bonds by a hydrolytic mechanism. Although comparative biochemical analyses have been published, no classification system has been proposed for HLDs, to date, that reconciles their phylogenetic and functional relationships. In the study presented here, we have analyzed all sequences and structures of genuine HLDs and their homologs detectable by database searches. Phylogenetic analyses revealed that the HLD family can be divided into three subfamilies denoted HLD-I, HLD-II, and HLD-III, of which HLD-I and HLD-III are predicted to be sister-groups. A mismatch between the HLD protein tree and the tree of species, as well as the presence of more than one HLD gene in a few genomes, suggest that horizontal gene transfers, and perhaps also multiple gene duplications and losses have been involved in the evolution of this family. Most of the biochemically characterized HLDs are found in the HLD-II subfamily. The dehalogenating activity of two members of the newly identified HLD-III subfamily has only recently been confirmed, in a study motivated by this phylogenetic analysis. A novel type of the catalytic pentad (Asp-His-Asp+Asn-Trp) was predicted for members of the HLD-III subfamily. Calculation of the evolutionary rates and lineage-specific innovations revealed a common conserved core as well as a set of residues that characterizes each HLD subfamily. The N-terminal part of the cap domain is one of the most variable regions within the whole family as well as within individual subfamilies, and serves as a preferential site for the location of relatively long insertions. The highest variability of discrete sites was observed among residues that are structural components of the access channels. Mutations at these sites modify the anatomy of the channels, which are important for the exchange of ligands between the buried active site and the bulk solvent, thus creating a structural basis for the molecular evolution of new substrate specificities. Our analysis sheds light on the evolutionary history of HLDs and provides a structural framework for designing enzymes with new specificities.
ESTHER : Chovancova_2007_Proteins_67_305
PubMedSearch : Chovancova_2007_Proteins_67_305
PubMedID: 17295320

Title : CAVER: a new tool to explore routes from protein clefts, pockets and cavities - Petrek_2006_BMC.Bioinformatics_7_316
Author(s) : Petrek M , Otyepka M , Banas P , Kosinova P , Koca J , Damborsky J
Ref : BMC Bioinformatics , 7 :316 , 2006
Abstract : BACKGROUND: The main aim of this study was to develop and implement an algorithm for the rapid, accurate and automated identification of paths leading from buried protein clefts, pockets and cavities in dynamic and static protein structures to the outside solvent. RESULTS: The algorithm to perform a skeleton search was based on a reciprocal distance function grid that was developed and implemented for the CAVER program. The program identifies and visualizes routes from the interior of the protein to the bulk solvent. CAVER was primarily developed for proteins, but the algorithm is sufficiently robust to allow the analysis of any molecular system, including nucleic acids or inorganic material. Calculations can be performed using discrete structures from crystallographic analysis and NMR experiments as well as with trajectories from molecular dynamics simulations. The fully functional program is available as a stand-alone version and as plug-in for the molecular modeling program PyMol. Additionally, selected functions are accessible in an online version. CONCLUSION: The algorithm developed automatically finds the path from a starting point located within the interior of a protein. The algorithm is sufficiently rapid and robust to enable routine analysis of molecular dynamics trajectories containing thousands of snapshots. The algorithm is based on reciprocal metrics and provides an easy method to find a centerline, i.e. the spine, of complicated objects such as a protein tunnel. It can also be applied to many other molecules. CAVER is freely available from the web site http://loschmidt.chemi.muni.cz/caver/.
ESTHER : Petrek_2006_BMC.Bioinformatics_7_316
PubMedSearch : Petrek_2006_BMC.Bioinformatics_7_316
PubMedID: 16792811

Title : Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris - Nakamura_2006_Protein.Expr.Purif_46_85
Author(s) : Nakamura T , Zamocky M , Zdrahal Z , Chaloupkova R , Monincova M , Prokop Z , Nagata Y , Damborsky J
Ref : Protein Expr Purif , 46 :85 , 2006
Abstract : Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.
ESTHER : Nakamura_2006_Protein.Expr.Purif_46_85
PubMedSearch : Nakamura_2006_Protein.Expr.Purif_46_85
PubMedID: 16216524

Title : Enzymes fight chemical weapons -
Author(s) : Prokop Z , Oplustil F , DeFrank J , Damborsky J
Ref : Biotechnol J , 1 :1370 , 2006
PubMedID: 17136732

Title : Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling - Banas_2006_J.Comput.Aided.Mol.Des_20_375
Author(s) : Banas P , Otyepka M , Jerabek P , Petrek M , Damborsky J
Ref : J Comput Aided Mol Des , 20 :375 , 2006
Abstract : 1,2,3-Trichloropropane (TCP) is a highly toxic, recalcitrant byproduct of epichlorohydrin manufacture. Haloalkane dehalogenase (DhaA) from Rhodococcus sp. hydrolyses the carbon-halogen bond in various halogenated compounds including TCP, but with low efficiency (k (cat)/K (m )= 36 s(-1) M(-1)). A Cys176Tyr-DhaA mutant with a threefold higher catalytic efficiency for TCP dehalogenation has been previously obtained by error-prone PCR. We have used molecular simulations and quantum mechanical calculations to elucidate the molecular mechanisms involved in the improved catalysis of the mutant, and enantioselectivity of DhaA toward TCP. The Cys176Tyr mutation modifies the protein access and export routes. Substitution of the Cys residue by the bulkier Tyr narrows the upper tunnel, making the second tunnel "slot" the preferred route. TCP can adopt two major orientations in the DhaA enzyme, in one of which the halide-stabilizing residue Asn41 forms a hydrogen bond with the terminal halogen atom of the TCP molecule, while in the other it bonds with the central halogen atom. The differences in these binding patterns explain the preferential formation of the (R)- over the (S)-enantiomer of 2,3-dichloropropane-1-ol in the reaction catalyzed by the enzyme.
ESTHER : Banas_2006_J.Comput.Aided.Mol.Des_20_375
PubMedSearch : Banas_2006_J.Comput.Aided.Mol.Des_20_375
PubMedID: 17016745

Title : Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities - Sato_2005_Appl.Environ.Microbiol_71_4372
Author(s) : Sato Y , Monincova M , Chaloupkova R , Prokop Z , Ohtsubo Y , Minamisawa K , Tsuda M , Damborsky J , Nagata Y
Ref : Applied Environmental Microbiology , 71 :4372 , 2005
Abstract : Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.
ESTHER : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedSearch : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedID: 16085827

Title : Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 - Kmunicek_2005_Biochemistry_44_3390
Author(s) : Kmunicek J , Hynkova K , Jedlicka T , Nagata Y , Negri A , Gago F , Wade RC , Damborsky J
Ref : Biochemistry , 44 :3390 , 2005
Abstract : Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants K(m) and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure-activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) second-shell residues Pro39 and Phe273. The tunnel and the second-shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.
ESTHER : Kmunicek_2005_Biochemistry_44_3390
PubMedSearch : Kmunicek_2005_Biochemistry_44_3390
PubMedID: 15736949
Gene_locus related to this paper: sphpi-linb

Title : Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases - Jesenska_2005_Appl.Environ.Microbiol_71_6736
Author(s) : Jesenska A , Pavlova M , Strouhal M , Chaloupkova R , Tesinska I , Monincova M , Prokop Z , Bartos M , Pavlik I , Rychlik I , Mobius P , Nagata Y , Damborsky J
Ref : Applied Environmental Microbiology , 71 :6736 , 2005
Abstract : Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.
ESTHER : Jesenska_2005_Appl.Environ.Microbiol_71_6736
PubMedSearch : Jesenska_2005_Appl.Environ.Microbiol_71_6736
PubMedID: 16269704
Gene_locus related to this paper: myctu-linb

Title : Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 - Nagata_2005_Appl.Environ.Microbiol_71_2183
Author(s) : Nagata Y , Prokop Z , Sato Y , Jerabek P , Kumar A , Ohtsubo Y , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 71 :2183 , 2005
Abstract : Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.
ESTHER : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedSearch : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedID: 15812056

Title : Crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 A resolution: dynamics of catalytic residues - Oakley_2004_Biochemistry_43_870
Author(s) : Oakley AJ , Klvana M , Otyepka M , Nagata Y , Wilce MC , Damborsky J
Ref : Biochemistry , 43 :870 , 2004
Abstract : We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C(alpha)-C(beta)-C(gamma) angle (126 degrees ) that deviated by 13.4 degrees from the "ideal" bond angle of 112.6 degrees. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C(alpha)-C(beta)-C(gamma) angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N(delta)(1) and Asp108 in conformation A gave the most exothermic reaction (DeltaH = -22 kcal/mol). With His272 doubly protonated at N(delta)(1) and N(epsilon)(2), the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C(alpha)-C(beta)-C(gamma) angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.
ESTHER : Oakley_2004_Biochemistry_43_870
PubMedSearch : Oakley_2004_Biochemistry_43_870
PubMedID: 14744129
Gene_locus related to this paper: sphpi-linb

Title : Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel - Chaloupkova_2003_J.Biol.Chem_278_52622
Author(s) : Chaloupkova R , Sykorova J , Prokop Z , Jesenska A , Monincova M , Pavlova M , Tsuda M , Nagata Y , Damborsky J
Ref : Journal of Biological Chemistry , 278 :52622 , 2003
Abstract : Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
ESTHER : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedSearch : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedID: 14525993

Title : Catalytic mechanism of the maloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 - Prokop_2003_J.Biol.Chem_278_45094
Author(s) : Prokop Z , Monincova M , Chaloupkova R , Klvana M , Nagata Y , Janssen DB , Damborsky J
Ref : Journal of Biological Chemistry , 278 :45094 , 2003
Abstract : 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.
ESTHER : Prokop_2003_J.Biol.Chem_278_45094
PubMedSearch : Prokop_2003_J.Biol.Chem_278_45094
PubMedID: 12952988
Gene_locus related to this paper: sphpi-linb

Title : Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates - Streltsov_2003_Biochemistry_42_10104
Author(s) : Streltsov VA , Prokop Z , Damborsky J , Nagata Y , Oakley A , Wilce MC
Ref : Biochemistry , 42 :10104 , 2003
Abstract : The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.
ESTHER : Streltsov_2003_Biochemistry_42_10104
PubMedSearch : Streltsov_2003_Biochemistry_42_10104
PubMedID: 12939138
Gene_locus related to this paper: sphpi-linb

Title : Comparative binding energy analysis of haloalkane dehalogenase substrates: modelling of enzyme-substrate complexes by molecular docking and quantum mechanical calculations - Kmunicek_2003_J.Comput.Aided.Mol.Des_17_299
Author(s) : Kmunicek J , Bohac M , Luengo S , Gago F , Wade RC , Damborsky J
Ref : J Comput Aided Mol Des , 17 :299 , 2003
Abstract : We evaluate the applicability of automated molecular docking techniques and quantum mechanical calculations to the construction of a set of structures of enzyme-substrate complexes for use in Comparative binding energy (COMBINE) analysis to obtain 3D structure-activity relationships. The data set studied consists of the complexes of eighteen substrates docked within the active site of haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10. The results of the COMBINE analysis are compared with previously reported data obtained for the same dataset from modelled complexes that were based on an experimentally determined structure of the DhlA-dichloroethane complex. The quality of fit and the internal predictive power of the two COMBINE models are comparable, but better external predictions are obtained with the new approach. Both models show a similar composition of the principal components. Small differences in the relative contributions that are assigned to important residues for explaining binding affinity differences can be directly linked to structural differences in the modelled enzyme-substrate complexes: (i) rotation of all substrates in the active site about their longitudinal axis, (ii) repositioning of the ring of epihalohydrines and the halogen substituents of 1,2-dihalopropanes, and (iii) altered conformation of the long-chain molecules (halobutanes and halohexanes). For external validation, both a novel substrate not included in the training series and two different mutant proteins were used. The results obtained can be useful in the future to guide the rational engineering of substrate specificity in DhlA and other related enzymes.
ESTHER : Kmunicek_2003_J.Comput.Aided.Mol.Des_17_299
PubMedSearch : Kmunicek_2003_J.Comput.Aided.Mol.Des_17_299
PubMedID: 14635723

Title : Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB - Nagata_2003_Appl.Environ.Microbiol_69_2349
Author(s) : Nagata Y , Prokop Z , Marvanova S , Sykorova J , Monincova M , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 69 :2349 , 2003
Abstract : The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.
ESTHER : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedSearch : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedID: 12676719

Title : Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene - Bosma_2002_Appl.Environ.Microbiol_68_3582
Author(s) : Bosma T , Damborsky J , Stucki G , Janssen DB
Ref : Applied Environmental Microbiology , 68 :3582 , 2002
Abstract : Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.
ESTHER : Bosma_2002_Appl.Environ.Microbiol_68_3582
PubMedSearch : Bosma_2002_Appl.Environ.Microbiol_68_3582
PubMedID: 12089046
Gene_locus related to this paper: rhoso-halo1

Title : Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains - Otyepka_2002_Protein.Sci_11_1206
Author(s) : Otyepka M , Damborsky J
Ref : Protein Science , 11 :1206 , 2002
Abstract : One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the alpha4'-helix-loop-alpha4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.
ESTHER : Otyepka_2002_Protein.Sci_11_1206
PubMedSearch : Otyepka_2002_Protein.Sci_11_1206
PubMedID: 11967377

Title : Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis - Bohac_2002_Biochemistry_41_14272
Author(s) : Bohac M , Nagata Y , Prokop Z , Prokop M , Monincova M , Tsuda M , Koca J , Damborsky J
Ref : Biochemistry , 41 :14272 , 2002
Abstract : Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.
ESTHER : Bohac_2002_Biochemistry_41_14272
PubMedSearch : Bohac_2002_Biochemistry_41_14272
PubMedID: 12450392
Gene_locus related to this paper: sphpi-linb

Title : Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26: evidence for product- and water-mediated inhibition - Oakley_2002_Biochemistry_41_4847
Author(s) : Oakley AJ , Prokop Z , Bohac M , Kmunicek J , Jedlicka T , Monincova M , Kuta-Smatanova I , Nagata Y , Damborsky J , Wilce MC
Ref : Biochemistry , 41 :4847 , 2002
Abstract : The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.
ESTHER : Oakley_2002_Biochemistry_41_4847
PubMedSearch : Oakley_2002_Biochemistry_41_4847
PubMedID: 11939779
Gene_locus related to this paper: sphpi-linb

Title : Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA - Jesenska_2002_Appl.Environ.Microbiol_68_3724
Author(s) : Jesenska A , Bartos M , Czernekova V , Rychlik I , Pavlik I , Damborsky J
Ref : Applied Environmental Microbiology , 68 :3724 , 2002
Abstract : Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.
ESTHER : Jesenska_2002_Appl.Environ.Microbiol_68_3724
PubMedSearch : Jesenska_2002_Appl.Environ.Microbiol_68_3724
PubMedID: 12147465
Gene_locus related to this paper: mycav-DHMA

Title : Structure-specificity relationships for haloalkane dehalogenases - Damborsky_2001_Environ.Toxicol.Chem_20_2681
Author(s) : Damborsky J , Rorije E , Jesenska A , Nagata Y , Klopman G , Peijnenburg WJ
Ref : Environ Toxicol Chem , 20 :2681 , 2001
Abstract : A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology. This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes. The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates). The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26). Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates. Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally. Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set). Correct predictions were obtained for 28 of 30 compounds in the validation set. Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated. A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases.
ESTHER : Damborsky_2001_Environ.Toxicol.Chem_20_2681
PubMedSearch : Damborsky_2001_Environ.Toxicol.Chem_20_2681
PubMedID: 11764149

Title : Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay: characterization of the haloalkane dehalogenase mutants - Marvanova_2001_J.Microbiol.Methods_44_149
Author(s) : Marvanova S , Nagata Y , Wimmerova M , Sykorova J , Hynkova K , Damborsky J
Ref : J Microbiol Methods , 44 :149 , 2001
Abstract : The pH indicator dye-based colorimetric method and multivariate experimental design were used for the systematic biochemical characterization of the broad-specificity enzymes haloalkane dehalogenases. Halogenated compounds for characterization of the enzymes were selected using Principal Component Analysis. The substrates were characterised by 24 physico-chemical and structural descriptors. Thirty-four substrates were selected for testing out of 194 halogenated compounds. Relative activities determined using the optimised colorimetric microplate assay were validated against the catalytic constants determined by gas chromatography. The applicability of the assay was tested with F151L, F154L and F169L mutants of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.
ESTHER : Marvanova_2001_J.Microbiol.Methods_44_149
PubMedSearch : Marvanova_2001_J.Microbiol.Methods_44_149
PubMedID: 11165344

Title : Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 - Kmunicek_2001_Biochemistry_40_8905
Author(s) : Kmunicek J , Luengo S , Gago F , Ortiz AR , Wade RC , Damborsky J
Ref : Biochemistry , 40 :8905 , 2001
Abstract : Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.
ESTHER : Kmunicek_2001_Biochemistry_40_8905
PubMedSearch : Kmunicek_2001_Biochemistry_40_8905
PubMedID: 11467952
Gene_locus related to this paper: xanau-halo1

Title : Effect of the carbon source on assessment of degrading bacteria with the spread-plating technique during in situ bioremediation - Damborsky_2000_Folia.Microbiol.(Praha)_45_35
Author(s) : Damborsky J , Damborska M , Stipek S , Jesenska A , Trantirek L , Sklenar V
Ref : Folia Microbiol (Praha) , 45 :35 , 2000
Abstract : Spread-plating belongs to traditional microbiological methods employed for quantification of subsurface microflora during bioremediation projects in the Czechia. Concentration of degrading organisms is estimated from the number of colonies grown on agar plates supplied with contaminant as the sole carbon source. The data obtained during in situ bioremediation of the Dacice site contaminated by cutting oil suggests that changes in the composition of the carbon source in the subsurface may cause a discrepancy between laboratory data and situation in subsurface.
ESTHER : Damborsky_2000_Folia.Microbiol.(Praha)_45_35
PubMedSearch : Damborsky_2000_Folia.Microbiol.(Praha)_45_35
PubMedID: 11200669

Title : Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria - Jesenska_2000_Appl.Environ.Microbiol_66_219
Author(s) : Jesenska A , Sedlacek I , Damborsky J
Ref : Applied Environmental Microbiology , 66 :219 , 2000
Abstract : Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.
ESTHER : Jesenska_2000_Appl.Environ.Microbiol_66_219
PubMedSearch : Jesenska_2000_Appl.Environ.Microbiol_66_219
PubMedID: 10618227

Title : Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 - Marek_2000_Biochemistry_39_14082
Author(s) : Marek J , Vevodova J , Smatanova IK , Nagata Y , Svensson LA , Newman J , Takagi M , Damborsky J
Ref : Biochemistry , 39 :14082 , 2000
Abstract : The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.
ESTHER : Marek_2000_Biochemistry_39_14082
PubMedSearch : Marek_2000_Biochemistry_39_14082
PubMedID: 11087355
Gene_locus related to this paper: sphpi-linb

Title : Determination of haloalkane dehalogenase activity by capillary zone electrophoresis - Glatz_2000_J.Chromatogr.A_895_219
Author(s) : Glatz Z , Marini MV , Wimmerova M , Damborsky J , Nagata Y
Ref : Journal of Chromatography A , 895 :219 , 2000
Abstract : A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.
ESTHER : Glatz_2000_J.Chromatogr.A_895_219
PubMedSearch : Glatz_2000_J.Chromatogr.A_895_219
PubMedID: 11105865

Title : Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 - Hynkova_1999_FEBS.Lett_446_177
Author(s) : Hynkova K , Nagata Y , Takagi M , Damborsky J
Ref : FEBS Letters , 446 :177 , 1999
Abstract : The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the gamma-hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl-enzyme intermediate. LinB is believed to belong to the family of alpha/beta-hydrolases which employ a catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the beta-strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the beta-strand seven.
ESTHER : Hynkova_1999_FEBS.Lett_446_177
PubMedSearch : Hynkova_1999_FEBS.Lett_446_177
PubMedID: 10100638
Gene_locus related to this paper: sphpi-linb

Title : Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons - Damborsky_1999_Protein.Eng_12_989
Author(s) : Damborsky J , Koca J
Ref : Protein Engineering , 12 :989 , 1999
Abstract : Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/beta-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments.
ESTHER : Damborsky_1999_Protein.Eng_12_989
PubMedSearch : Damborsky_1999_Protein.Eng_12_989
PubMedID: 10585505

Title : Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26 - Nagata_1999_Protein.Expr.Purif_17_299
Author(s) : Nagata Y , Hynkova K , Damborsky J , Takagi M
Ref : Protein Expr Purif , 17 :299 , 1999
Abstract : The linB gene product (LinB), which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of haloalkane dehalogenases with a broad range of substrate specificity. Elucidation of the factors determining its substrate specificity is of interest. Aiming to facilitate purification of recombinant LinB protein for site-directed mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB. The His-tagged LinB was specifically bound with Ni-NTA resin in the buffer containing 10 mM imidazole. After elution with 500 mM imidazole, quantitative recovery of protein occurred. The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB. Although the His-tagged LinB expressed in an average of 80% of the activity of the wild type LinB for 10 different substrates, the decrease was very similar for different substrates with the standard deviation of 5.5%. The small activity reduction is independent of the substrate shape, size, or number of substituents, indicating that the His-tagged LinB can be used for further mutagenesis studies. To confirm the suitability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were constructed, purified, and tested for activity. As expected, complete loss in activity of W109L and sustained activity of F151W were observed.
ESTHER : Nagata_1999_Protein.Expr.Purif_17_299
PubMedSearch : Nagata_1999_Protein.Expr.Purif_17_299
PubMedID: 10545279

Title : Computational site-directed mutagenesis of haloalkane dehalogenase in position 172 - Damborsky_1998_Protein.Eng_11_901
Author(s) : Damborsky J , Bohac M , Prokop M , Kuty M , Koca J
Ref : Protein Engineering , 11 :901 , 1998
Abstract : The application of molecular modelling and quantum-chemistry calculations for the 'computational site-directed mutagenesis' of haloalkane dehalogenase is described here. The exhaustive set of single point mutants of haloalkane dehalogenase in position 172 was constructed by homology modelling. The ability of substituting residues to stabilize the halide ion formed during the dehalogenation reaction in the enzyme active site was probed by quantum-chemical calculations. A simplified modelling procedure was adopted to obtain informative results on the potential activity of mutant proteins in a sufficiently short period of time, which, in the future, could be applicable for making bona fide predictions of mutants' activity prior to their preparation in the laboratory. The reaction pathways for the carbon-halide bond cleavage were calculated using microscopic models of wild type and mutant proteins. The theoretical parameters derived from the calculation, i.e. relative energies and selected atomic charges of educt, product and transition state structures, were statistically correlated with experimentally determined activities. The charge difference of educt and product on the halide-stabilizing hydrogen atom of residue 172 was the best parameter to distinguish protein variants with high activity from mutant proteins displaying a low activity. All mutants with significant activity in the experiment were found to have this parameter one order of magnitude higher than mutants with low activity. The results obtained are discussed in the light of the practical application of this methodology for the prediction of potentially active protein variants. Further automation of the modelling procedure is suggested for combinatorial screening of the large number of protein variants. Coupling of the dehalogenation reaction with hydrogenation of the halide ion formed during the reaction in the enzyme active site was proposed as a possible way to improve the catalytic activity of the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10.
ESTHER : Damborsky_1998_Protein.Eng_11_901
PubMedSearch : Damborsky_1998_Protein.Eng_11_901
PubMedID: 9862209

Title : Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26 - Nagata_1997_Appl.Environ.Microbiol_63_3707
Author(s) : Nagata Y , Miyauchi K , Damborsky J , Manova K , Ansorgova A , Takagi M
Ref : Applied Environmental Microbiology , 63 :3707 , 1997
Abstract : The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
ESTHER : Nagata_1997_Appl.Environ.Microbiol_63_3707
PubMedSearch : Nagata_1997_Appl.Environ.Microbiol_63_3707
PubMedID: 9293022
Gene_locus related to this paper: sphpi-linb

Title : Repositioning the catalytic triad aspartic acid of haloalkane dehalogenase: effects on stability, kinetics, and structure - Krooshof_1997_Biochemistry_36_9571
Author(s) : Krooshof GH , Kwant EM , Damborsky J , Koca J , Janssen DB
Ref : Biochemistry , 36 :9571 , 1997
Abstract : Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other alpha/beta-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.
ESTHER : Krooshof_1997_Biochemistry_36_9571
PubMedSearch : Krooshof_1997_Biochemistry_36_9571
PubMedID: 9236003

Title : Some biochemical properties and the classification of a range of bacterial haloalkane dehalogenases - Damborsky_1997_Biotechnol.Appl.Biochem_26_19
Author(s) : Damborsky J , Nyandoroh MG , Nemec M , Holoubek I , Bull AT , Hardman DJ
Ref : Biotechnol Appl Biochem , 26 :19 , 1997
Abstract : Multivariate analyses and experimental data have been used to evaluate the relationships between eight bacterial hydrolytic haloalkane dehalogenases. The results indicate that seven of the dehalogenases investigated can confidently be placed into two Classes [sensu Slater, Bull and Hardman (1995) Biodegradation 6, 181-189] according to their substrate profiles. The remaining enzyme, isolated from Rhodococcus erythropolis CP9, appears to represent a third Class of haloalkane dehalogenases.
ESTHER : Damborsky_1997_Biotechnol.Appl.Biochem_26_19
PubMedSearch : Damborsky_1997_Biotechnol.Appl.Biochem_26_19
PubMedID: 9261999

Title : A mechanistic approach to deriving quantitative structure-activity relationship models for microbial degradation of organic compounds - Damborsky_1996_SAR.QSAR.Environ.Res_5_27
Author(s) : Damborsky J
Ref : SAR QSAR Environ Research , 5 :27 , 1996
Abstract : Quantitative Structure-Biodegradability Relationships (QSBRs), relate the molecular structure of an organic chemical to its biological degradability. The high complexity of the microbial degradation process as well as great variety of the interactions between organism, organic pollutant and the environment make it difficult to interpret the results from biodegradability experiments. Literature concerning the various approaches applied in Structure-Biodegradability Relationship modeling is reviewed and the reliability and applicability of the results obtained from different biodegradation tests is discussed. A mechanistic approach, based on comparison of the data measured at different organization levels, will hopefully contribute to a better understanding of mechanisms taking place during the biodegradation. Their description and quantification may lead to improvement of existing QSBR models.
ESTHER : Damborsky_1996_SAR.QSAR.Environ.Res_5_27
PubMedSearch : Damborsky_1996_SAR.QSAR.Environ.Res_5_27
PubMedID: 8640583