Bednar D

References (29)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Title : Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17 - Chrast_2019_Microorganisms_7_
Author(s) : Chrast L , Tratsiak K , Planas-Iglesias J , Daniel L , Prudnikova T , Brezovsky J , Bednar D , Kuta Smatanova I , Chaloupkova R , Damborsky J
Ref : Microorganisms , 7 : , 2019
Abstract : Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 degrees C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 A resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.
ESTHER : Chrast_2019_Microorganisms_7_
PubMedSearch : Chrast_2019_Microorganisms_7_
PubMedID: 31661858
Gene_locus related to this paper: 9alte-a3jb27

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

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

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

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