Hollfelder F

References (15)

Title : Ultrahigh-throughput directed evolution of a metal-free alpha\/beta-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase - Schnettler-Fernandez_2022_Biorxiv__
Author(s) : Schnettler-Fernandez D , Klein OJ , Kaminski TS , Colin PY , Hollfelder F
Ref : Biorxiv , : , 2022
Abstract : The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators. Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the alpha/beta-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105 members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91's phosphotriesterase activity was increased ~ 400-fold to a kcat/KM of ~ 10 6 M-1 s-1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in Nature, resulting in alternative reagents with a novel catalytic machinery.
ESTHER : Schnettler-Fernandez_2022_Biorxiv__
PubMedSearch : Schnettler-Fernandez_2022_Biorxiv__
PubMedID:
Gene_locus related to this paper: 9bact-KP212148

Title : Ultrahigh-Throughput Directed Evolution of a Metal-Free alpha\/beta-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase - Schnettler_2022_J.Am.Chem.Soc__
Author(s) : Schnettler JD , Klein OJ , Kaminski TS , Colin PY , Hollfelder F
Ref : Journal of the American Chemical Society , : , 2022
Abstract : Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of =360 (to a k(cat)/K(M) of =7 x 10(5) M(-1) s(-1)) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.
ESTHER : Schnettler_2022_J.Am.Chem.Soc__
PubMedSearch : Schnettler_2022_J.Am.Chem.Soc__
PubMedID: 36583539
Gene_locus related to this paper: 9bact-KP212148

Title : Adventures on the routes of protein evolution - in memoriam Dan Salah Tawfik (1955 - 2021) - Jackson_2022_J.Mol.Biol_434_167462
Author(s) : Jackson C , Toth-Petroczy A , Kolodny R , Hollfelder F , Fuxreiter M , Caroline Lynn Kamerlin S , Tokuriki N
Ref : Journal of Molecular Biology , :167462 , 2022
Abstract : Understanding how proteins evolved not only resolves mysteries of the past, but also helps address challenges of the future, particularly those relating to the design and engineering of new protein functions. Here we review the work of Dan S. Tawfik, one of the pioneers of this area, highlighting his seminal contributions in diverse fields such as protein design, high throughput screening, protein stability, fundamental enzyme-catalyzed reactions and promiscuity, that underpin biology and the origins of life. We discuss the influence of his work on how our models of enzyme and protein function have developed and how the main driving forces of molecular evolution were elucidated. The discovery of the rugged routes of evolution has enabled many practical applications, some which are now widely used.
ESTHER : Jackson_2022_J.Mol.Biol_434_167462
PubMedSearch : Jackson_2022_J.Mol.Biol_434_167462
PubMedID: 35104498

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 : 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 : 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 : Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase - van Loo_2019_Biochemistry_58_1363
Author(s) : van Loo B , Berry R , Boonyuen U , Mohamed MF , Golicnik M , Hengge AC , Hollfelder F
Ref : Biochemistry , 58 :1363 , 2019
Abstract : Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS(WT) has a much less negative Bronsted coefficient (beta(leavingsgroup)(obs-Enz) = -0.33) than the uncatalyzed reaction (beta(leavingsgroup)(obs) = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an (18)O(bridge) KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. (15)N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D(2)O (deltabeta(leavingsgroup)(H-D) = +0.06) suggest that the mechanism for S-O(bridge) bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. (18)O(nonbridge) KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).
ESTHER : van Loo_2019_Biochemistry_58_1363
PubMedSearch : van Loo_2019_Biochemistry_58_1363
PubMedID: 30810299

Title : Specificity Effects of Amino Acid Substitutions in Promiscuous Hydrolases: Context-Dependence of Catalytic Residue Contributions to Local Fitness Landscapes in Nearby Sequence Space - Bayer_2017_Chembiochem_18_1001
Author(s) : Bayer CD , van Loo B , Hollfelder F
Ref : Chembiochem , 18 :1001 , 2017
Abstract : Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects. Instead, these effects appear to be dominated by their structural context. Likewise, the effect of changing the catalytic nucleophile itself is not reaction-type-specific. Mapping of 'fitness landscapes' of four activities onto the possible variation of a chosen sequence position revealed tremendous potential for respecialization of AP superfamily members through single-point mutations, highlighting catalytic promiscuity as a powerful predictor of adaptive potential.
ESTHER : Bayer_2017_Chembiochem_18_1001
PubMedSearch : Bayer_2017_Chembiochem_18_1001
PubMedID: 28464395

Title : The role of protein dynamics in the evolution of new enzyme function - Campbell_2016_Nat.Chem.Biol_12_944
Author(s) : Campbell EC , Kaltenbach M , Correy GJ , Carr PD , Porebski BT , Livingstone EK , Afriat-Jurnou L , Buckle AM , Weik M , Hollfelder F , Tokuriki N , Jackson CJ
Ref : Nat Chemical Biology , 12 :944 , 2016
Abstract : Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
ESTHER : Campbell_2016_Nat.Chem.Biol_12_944
PubMedSearch : Campbell_2016_Nat.Chem.Biol_12_944
PubMedID: 27618189

Title : Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity - Kaltenbach_2016_PLoS.Genet_12_e1006305
Author(s) : Kaltenbach M , Emond S , Hollfelder F , Tokuriki N
Ref : PLoS Genet , 12 :e1006305 , 2016
Abstract : The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.
ESTHER : Kaltenbach_2016_PLoS.Genet_12_e1006305
PubMedSearch : Kaltenbach_2016_PLoS.Genet_12_e1006305
PubMedID: 27716796

Title : Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics - Colin_2015_Nat.Commun_6_10008
Author(s) : Colin PY , Kintses B , Gielen F , Miton CM , Fischer G , Mohamed MF , Hyvonen M , Morgavi DP , Janssen DB , Hollfelder F
Ref : Nat Commun , 6 :10008 , 2015
Abstract : Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.
ESTHER : Colin_2015_Nat.Commun_6_10008
PubMedSearch : Colin_2015_Nat.Commun_6_10008
PubMedID: 26639611
Gene_locus related to this paper: 9bact-KP212148

Title : Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease - Shafee_2015_Chembiochem_16_1866
Author(s) : Shafee T , Gatti-Lafranconi P , Minter R , Hollfelder F
Ref : Chembiochem , 16 :1866 , 2015
Abstract : Mutation of the tobacco etch virus (TEV) protease nucleophile from cysteine to serine causes an approximately -10(4) -fold loss in activity. Ten rounds of directed evolution of the mutant, TEV(Ser) , overcame the detrimental effects of nucleophile exchange to recover near-wild-type activity in the mutant TEV(Ser) X. Rather than respecialising TEV to the new nucleophile, all the enzymes along the evolutionary trajectory also retained the ability to use the original cysteine nucleophile. Therefore the adaptive evolution of TEV(Ser) is paralleled by a neutral trajectory for TEV(Cys) , in which mutations that increase serine nucleophile reactivity hardly affect the reactivity of cysteine. This apparent nucleophile permissiveness explains how nucleophile switches can occur in the phylogeny of the chymotrypsin-like protease PA superfamily. Despite the changed key component of their chemical mechanisms, the evolved variants TEV(Ser) X and TEV(Cys) X have similar activities; this could potentially facilitate escape from adaptive conflict to enable active-site evolution.
ESTHER : Shafee_2015_Chembiochem_16_1866
PubMedSearch : Shafee_2015_Chembiochem_16_1866
PubMedID: 26097079

Title : Evolution of enzyme catalysts caged in biomimetic gel-shell beads - Fischlechner_2014_Nat.Chem_6_791
Author(s) : Fischlechner M , Schaerli Y , Mohamed MF , Patil S , Abell C , Hollfelder F
Ref : Nat Chem , 6 :791 , 2014
Abstract : Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these man-made compartments can be identified readily by fluorescence-activated sorting at rates >10(7) GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components.
ESTHER : Fischlechner_2014_Nat.Chem_6_791
PubMedSearch : Fischlechner_2014_Nat.Chem_6_791
PubMedID: 25143214

Title : Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology - Theberge_2010_Angew.Chem.Int.Ed.Engl_49_5846
Author(s) : Theberge AB , Courtois F , Schaerli Y , Fischlechner M , Abell C , Hollfelder F , Huck WT
Ref : Angew Chem Int Ed Engl , 49 :5846 , 2010
Abstract : Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.
ESTHER : Theberge_2010_Angew.Chem.Int.Ed.Engl_49_5846
PubMedSearch : Theberge_2010_Angew.Chem.Int.Ed.Engl_49_5846
PubMedID: 20572214

Title : New genotype-phenotype linkages for directed evolution of functional proteins - Leemhuis_2005_Curr.Opin.Struct.Biol_15_472
Author(s) : Leemhuis H , Stein V , Griffiths AD , Hollfelder F
Ref : Current Opinion in Structural Biology , 15 :472 , 2005
Abstract : The key practical consideration in directed evolution of functional biomolecules is the linkage of genotype and phenotype. In vitro selections offer the potential to select from libraries with up to 10(10)-10(14) members, with fewer constraints than current cell-based selections. New approaches such as mRNA display, ribosome display and in vitro compartmentalisation have complementary areas of application in selections for binding or catalysis.
ESTHER : Leemhuis_2005_Curr.Opin.Struct.Biol_15_472
PubMedSearch : Leemhuis_2005_Curr.Opin.Struct.Biol_15_472
PubMedID: 16043338