Prokop Z

References (68)

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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB - Gross_2016_Chembiochem_17_1437
Author(s) : Gross J , Prokop Z , Janssen D , Faber K , Hall M
Ref : Chembiochem , 17 :1437 , 2016
Abstract : The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product.
ESTHER : Gross_2016_Chembiochem_17_1437
PubMedSearch : Gross_2016_Chembiochem_17_1437
PubMedID: 27223496

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

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

Title : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : 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