Article

Computerbasiertes Enzymdesign

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  • Biotech Startup (in stealth mode)
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Abstract

Der “Inside-Out”-Ansatz des computerbasierten Enzymdesigns vereint die neuesten Entwicklungen im Bereich der Computerchemie und -biologie. In diesem Rahmen ist es möglich geworden, Proteine zur Katalyse von Reaktionen herzustellen, für die es in der Natur kein Pendant gibt. Die Erfolgsrate ist gegenwärtig gering, sodass nur ein Bruchteil dieser Konzeptproteine zum Schluss wie geplant funktioniert. Errungenschaften, aber auch Beschränkungen der gegenwärtigen Technologie werden hier behandelt und mit anderen Methoden verglichen. Auf sich alleine gestellt, ermöglicht der “Inside-Out”-Ansatz die Produktion von Proteinen mit katalytischer Aktivität und Selektivität – wenn auch mit bescheidenen kinetische Eigenschaften im Vergleich zu natürlich vorkommenden Enzymen. Gerichtete Evolution, Proteindynamiksimulationen und Crowd-Sourcing können jedoch zu erheblich verbesserten Varianten solcher “Inside-Out”-Proteine führen.

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Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Hohe Selektivitäten und ausgezeichnete Kontrolle des Reaktionsverlaufs regen Chemiker dazu an, in der organischen Synthese Biokatalysatoren einzusetzen. Viele nützliche Reaktionen sind auf diese Weise jedoch nicht zugänglich, weil sie im Repertoire der Natur nicht vorkommen. In diesem Aufsatz werden wir einen evolutiven Ansatz zur Entwicklung von Enzymen beschreiben, die solche nicht-natürlichen Reaktionen katalysieren. Wir beginnen mit Beispielen dafür, wie die Natur neue katalytische Funktionen entdeckt hat und wie solche evolutiven Fortschritte im Labor nachvollzogen wurden, indem man von existierenden Enzymen ausging. Wir untersuchen dann nicht-natürliche Enzymaktivitäten, die für die chemische Synthese entdeckt und verwendet wurden, wobei wir uns auf Reaktionen ohne natürliches Pendant konzentrieren. Wir zeigen Beispiele, wie nicht-natürliche Aktivitäten durch gerichtete Evolution verbessert wurden, indem der Prozess nachgeahmt wurde, mit dem die Natur neue Katalysatoren erzeugt. Zum Schluss beschreiben wir noch Entdeckungen nicht-natürlicher katalytischer Funktionen, die künftig Chancen auf eine Ausdehnung des enzymatischen Universums bieten können.
Article
High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.
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Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.
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Thanks to substantial improvements in the theory of metabolic fluxes and the application of 13C isotope markers in experimental flux studies, Pareto efficiency of bacterial metabolism can now be determined and direct answers to the long standing questions of optimization according to multiple criteria in nature can be given. Cells or organisms operate close to Pareto optima but the performance with respect to every single criterion is almost always improvable. Rational design and evolutionary methods are routinely used for the production of biomolecules with optimized properties. Examples are proteins for technical applications, for example in detergents, and optimally binding nucleic acid molecules called aptamers. Among the various perspectives of synthetic biology, the usage of DNA for information storage is particularly promising: In a pilot experiment, an entire book including figures and a Java script, in total more than 5 megabit, were stored on a single DNA molecule. © 2013 Wiley Periodicals, Inc. Complexity 18: 21–31, 2013
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Einmal anders: In einer promiskuitiven Reaktion katalysiert das Enzym Cytochrom P450-BM3 die Cyclopropanierung von Olefinen, zum Teil mit hoher Stereoselektivität (siehe Schema). Diese Untersuchung demonstriert, dass das Design ungewöhnlicher Enzympromiskuität möglich ist.
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Präzises Design des aktiven Zentrums durch Protein‐Engineering führte zur Entwicklung einer hocheffizienten Kemp‐Eliminase (siehe Struktur mit Substrat in der Bindungstasche). Das Ausgangsprotein mit geringer Aktivität basierte auf Computerdesign, da kein natürliches Enzym mit dieser Aktivität bekannt war. Dies ist ein Durchbruch für das Proteindesign, da nun neue katalytische Aktivitäten greifbar sind, die die Effizienz natürlicher Enzymkatalyse erreichen.
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We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app, 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.
Article
Biocatalysis uses natural enzymes to perform a desired reaction, and numerous examples covering almost all enzyme classes are well-documented in the literature. If a natural enzyme does not show the desired performance, then methods of protein engineering[1] can be used to adapt its properties to meet a given target such as stereoselectivity, desired substrate scope, or stability to heat or organic solvents. Particularly over the past two decades, many success stories based on various tools of protein engineering-ranging from random mutagenesis combined with highthroughput screening to rational protein design methods-have been written. This also includes various industrial processes based on specifically designed biocatalysts[2].
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De novo designing of functional membrane proteins is fundamental in terms of understanding the structure, folding, and stability of membrane proteins. In this work, we report the design and characterization of a transmembrane protein, termed HETPRO (HEme-binding Transmembrane PROtein), that binds two molecules of heme in a membrane and catalyzes oxidation/reduction reactions. The primary structure of HETPRO has been optimized in a guided fashion, from an antimicrobial peptide, for transmembrane orientation, defined 3D structure, and functions. HETPRO assembles into a tetrameric form, from an apo dimeric helical structure, in complex with cofactor in detergent micelles. The NMR structure of the apo HETPRO in micelles reveals an antiparallel helical dimer that inserts into the nonpolar core of detergent micelles. The well-defined structure of HETPRO and its ability to bind to heme moieties could be utilized to develop a functional membrane protein mimic for electron transport and photosystems.
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A general strategy is described that integrates data mining of enzyme families and molecular modeling of enzyme–substrate interactions to identify selectivity hotspots and to design focused variant libraries for changed regio- and stereoselectivity. This strategy is demonstrated for two case studies; the design of cytochrome P450 monooxygenases with improved regioselectivity and of thiamine diphosphate dependent enzymes with improved stereoselectivity. In both families, two selectivity hotspots are found in almost all sequences, and simple, generic rules are established to predict the effect of mutations at these positions on selectivity. The crucial role of the hotspot positions is validated for an increasing number of enzymes by designing variants with improved or switched selectivity.
Article
RA110 is a computationally designed retro-aldolase enzyme that utilizes amine catalysis to convert 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. The original design accelerated substrate cleavage by a factor of 12 000 over background, and its activity was subsequently increased more than a thousand-fold by directed evolution. The X-ray structure of the evolved catalyst covalently modified with a 1,3-diketone inhibitor deviates substantially from the design model, however, with the ligand adopting a completely different orientation than predicted. Moreover, significant activity was maintained even after relocation of the reactive lysine within the apolar binding pocket. These results suggest that the success of the original design is not ascribable to atomically accurate molecular recognition, but rather to successful placement of a reactive lysine adjacent to an apolar binding pocket. Nevertheless, the stabilizing interactions observed at the active site of the evolved variant suggest that improvements in the precision of design calculations will afford enzymes with higher catalytic activities.
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How about this for a change: The promiscuous enzyme cytochrome P450-BM3 catalyzes the cyclopropanation of olefins, sometimes with high stereoselectivity. The highlighted paper demonstrates that it is possible to design unusual enzyme promiscuity.
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Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1–infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.
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This paper describes the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. These antibodies use the enamine mechanism common to the natural Class I aldolase enzymes. Substrates for these catalysts, 23 donors and 16 acceptors, have been identified. The aldol acceptor specificity is expected to be much broader than that defined here since all aldehydes tested, with the exception polyhydroxylated aldehydes, were substrates for the antibodies. 38C2 and 33F12 have been shown to catalyze intermolecular ketone−ketone, ketone−aldehyde, aldehyde−ketone, and aldehyde−aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions have also been defined. With acetone as the aldol donor substrate a new stereogenic center is formed by attack on the si-face of the aldehyde with ee's in most cases exceeding 95%. With hydroxyacetone as the donor substrate, attack occurs on the re-face, generating an α,β-dihydroxy ketone with two stereogenic centers of the α-syn configuration in 70 to >98% ee. With fluoroacetone donor reactions, the major product is a syn α-fluoro-β-hydroxy ketone with 95% ee. Studies of retroaldol reactions demonstrate that the antibodies provide up to 108-fold enhanced efficiency relative to simple amine-catalyzed reactions.
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This review considers the advances made in using computer simulations to elucidate the catalytic power of enzymes. It is shown that some current approaches, and in particular the empirical valence bond approach, allow us to describe enzymatic reactions by rigorous concepts of current chemical physics and to estimate any proposed catalytic contribution. This includes evaluation of activation free energies, nonequilibrium solvation, quantum mechanical tunneling, entropic effects, and other factors. The ability to evaluate activation free energies for reactions in water and proteins allows us to simulate the rate acceleration in enzymatic reactions. It is found that the most important contribution to catalysis comes from the reduction of the activation free energy by electrostatic effects. These effects are found to be associated with the preorganized polar environment of the enzyme active site. The use of computer simulations as effective tools for examining different catalytic proposals is illustrated by two examples. First, we consider the popular proposal that enzymes catalyze reactions by special dynamical effects. It is shown that this proposal is not supported by any consistent simulation study. It is also shown that the interpretation of recent experiments as evidence for dynamical contributions to catalysis is unjustified. Obviously, all chemical reactions involve motion, but unless this motion provides non-Boltzmann probability for reaching the transition state there are not dynamical effects. Vibrationally enhanced tunneling is shown to be a well understood phenomenon that does not lead to special catalytic effects. Similarly, it is shown that nonequilibrium solvation effects do not constitute dynamical contributions to catalysis. Second, the effectiveness of simulation approaches is also demonstrated in studies of entropic contributions to catalysis. It is found that the corresponding contributions are smaller than previously thought.
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Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K(m) values of ≤ 5 10(4) M(-1)s(-1)). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k(cat)/K(m), and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K(m) values up to 0.6 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of ≤ 10(7) almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.
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A series of eleven immunizations against transition-state analogs were carried out to improve the catalytic properties of Ab 9D9, a catalytic antibody that catalyzes a fluorogenic retro-Diels-Alder reaction liberating nitroxyl. By a direct fluorescence assay of cell-culture supernatant, eight new hybridoma cell lines producing catalytic antibodies for the reaction were readily identified among more than 14000 individual samples. Our results demonstrate that early catalysis screening by fluorescence allows an efficient survey of large antibody libraries, and may lead to rapid and significant improvement in catalysis.
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The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
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Die Affinitäten von Wirten – ausgehend von kleinen synthetischen Cavitanten bis hin zu großen Proteinen – für organische Moleküle sind gut dokumentiert. Die mittleren Assoziationskonstanten für die Bindung organischer Moleküle durch Cyclodextrine, synthetische Wirte und Albumine in Wasser sowie von katalytischen Antikörpern oder Enzymen für Substrate betragen im Allgemeinen 103.5±2.5 M−1. Die Bindungsaffinitäten steigen bei der Komplexierung von Übergangszuständen und biologischen Antigenen durch Antikörper oder von Enzymen durch Inhibitoren auf 108±2 M−1 und auf 1016±4 M−1 für Enzym-Übergangszustands-Komplexe. Die Gründe für die unterschiedlichen Stabilitäten dieser Wirt-Gast-Systeme sollen hier untersucht werden, und wir beschreiben Ansätze zur computergestützten Analyse der Wirt-Gast-Komplexbildung in Lösung. Bei vielen Komplexklassen besteht eine ungefähre Korrelation der Bindungsaffinität mit der Größe der Oberfläche, die bei der Komplexierung vergraben wird. Enzyme folgen dieser Korrelation nicht, sondern binden Übergangszustände sehr viel stärker als es anhand der Oberfläche zu erwarten wäre.
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Neun effiziente Aldolase-Antikörper wurden durch Verwendung des Haptens 1 hergestellt. Bei diesem Hapten sind die Konzepte der reaktiven Immunisierung und des Übergangszustandsanalogons in einem einzigen Molekül vereint. Die Charakterisierung von zwei solchen Antikörpern ergab, daß sie hocheffiziente (bis zu 1000fach wirksamer als alle bekannten katalytischen Antikörper) und enantioselektive Katalysatoren für Aldol- und Retro-Aldolreaktionen sind und umgekehrte Enantio- und Diastereoselektivitäten wie der Antikörper 38C2 aufweisen.
Article
Asymmetrische Katalyse spielt eine Schlüsselrolle in der modernen synthetischen organischen Chemie, wobei künstliche Katalysatoren und Enzyme die zwei wichtigsten Optionen darstellen. Die Verwendung von Enzymen in der organischen Chemie und Biotechnologie hat in der zweiten Hälfte des vergangenen Jahrhunderts stark zugenommen, oft blieb jedoch der Anwendungsbereich aus mehreren Gründen eingeschränkt. Dazu gehören begrenzte Substratakzeptanz, geringe oder fehlende Stereoselektivität, unzureichende Stabilität und manchmal Produktinhibierung. Während der vergangenen 15 Jahre wurde die genetische Technik der gerichteten Evolution so weit entwickelt und verfeinert, dass all diese Probleme angegangen und in der Regel gelöst werden können. Sie basiert auf wiederholende Zyklen von Genmutagenese, Expression und Screening (oder Selektion). Der vorliegende Aufsatz fokussiert auf gerichtete Evolution stereoselektiver Enzyme, ein fundamental neuer Ansatz zur asymmetrischen Katalyse. Der Schwerpunkt liegt auf Methodenentwicklung in dem Bestreben, diese Art der Katalysator-Optimierung einfacher, schneller und effizienter als in der Vergangenheit zu gestalten. Die neueren Methoden bescheren dem Chemiker und dem Biotechnologen robuste und selektive Katalysatoren für eine Vielzahl von nützlichen Anwendungen.
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Antibody 21D8 catalyzes the solvent-sensitive decarboxylation of 3-carboxybenzisoxazoles. The crystal structure of chimeric Fab 21D8 with and without hapten at 1.61 A and 2.10 A, respectively, together with computational analysis, shows how a melange of polar and non-polar sites are exploited to achieve both substrate binding and acceleration of a reaction normally facilitated by purely aprotic dipolar media. The striking similarity of the decarboxylase and a series of unrelated esterase antibodies also highlights the chemical versatility of structurally conserved anion binding sites and the relatively subtle changes involved in fine-tuning the immunoglobulin pocket for recognition of different ligands and catalysis of different reactions.
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Studies of catalytic antibodies have provided examples of mechanisms ranging from simple transition-state stabilization to multi-step pathways involving covalent intermediates. This review critically compares catalytic antibodies with other biological catalysts, using amide and ester hydrolysis as examples. By means of kinetic simulations, the catalytic efficiencies of many of these antibodies are shown to be limited by a number of factors including relatively weak substrate recognition coupled with relatively strong product binding. This analysis also suggests specific methods to improve catalytic antibodies, either through more sophisticated transition-state analogue design or by mutagenesis of existing antibodies.
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We assume that each class of protein has a core structure that is defined by internal residues, and that the external, solvent-contacting residues contribute to the stability of the structure, are of primary importance to function, but do not determine the architecture of the core portions of the polypeptide chain. An algorithm has been developed to supply a list of permitted sequences of internal residues compatible with a known core structure. This list is referred to as the tertiary template for that structure. In general the positions in the template are not sequentially adjacent and are distributed throughout the polypeptide chain. The template is derived using the fixed positions for the main-chain and beta-carbon atoms in the test structure and selected stereochemical rules. The focus of this paper is on the use of two packing criteria: avoidance of steric overlap and complete filling of available space. The program also notes potential polar group interactions and disulfide bonds as well as possible burial of formal charges.
Article
The crystal structure of an efficient Diels-Alder antibody catalyst at 1.9 angstrom resolution reveals almost perfect shape complementarity with its transition state analog. Comparison with highly related progesterone and Diels-Alderase antibodies that arose from the same primordial germ line template shows the relatively subtle mutational steps that were able to evolve both structural complementarity and catalytic efficiency.
Article
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity towards immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a kcat/KM of 568 M-1s-1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases, and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal, and redefining its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.
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The mechanism of reaction of 5-, 6-, and 7-substituted benzisoxazoles with hydroxide or amines has been established as a concerted E2 elimination yielding o-cyanophenolate anions. With hydroxide in ethanol-water mixtures, these reactions have been shown to have a ΔH° in the range of -35 to -39 kcal/mol. The effects of salts and temperature on rates are considered, and the significance of the benzisoxazole system as a new kind of leaving group is discussed.
Article
A complex of the five-membered transition structure for intramolecular cyclization of 4,5-epoxyhexan-1-ol with the “Houk” theozyme which is 3.5 kcal/mol lower in energy than the complex previously reported as a model for the antibody IgG26D9 catalyzed intramolecular cyclization reverses the preference of that theozyme to favor furan formation. This shows the reported theozyme complex was not a global minima and therefore not a satisfactory model for the antibody reaction. We report a new theozyme favoring pyran formation consistent with the antibody result and the recently published crystal structure of the antibody Fab 5C8 with hapten.
Article
An iterative redesign protocol for the transformation of a non-native peptide into a series of nativelike proteins derived from elementary considerations of biological evolution coupled with 1H NMR as an artificial selection criterion is presented. Each of three heptad d position leucines in the helix−helix interfaces of the prototype heme protein maquette, [H10H24]2 or (α-SS-α)2, were replaced in a unit modification per helix by more conformationally restricted β-branched and aromatic amino acids. The secondary structure content (evaluated by circular dichroism and infrared spectroscopies), solvent accessibility of the tryptophan residues (measured by fluorescence spectroscopy), global stability (quantitated by isothermal chemical denaturation), and degree of conformational specificity (determined by 1H NMR spectroscopy) of the resultant peptides were determined. Improvement in the degree of conformational specificity was utilized as a selection criterion to choose three of the nine singly modified peptides for a second unit modification per helix. Five of the resultant seven doubly modified peptides were nativelike, as determined by NMR spectroscopy. One of the doubly modified peptides was chosen for a third unit modification per helix, which resulted in three triple variants with low conformational specificity. The 20 proteins synthesized fold into discrete, stable four-α-helix bundles but with differing stabilities (−ΔGH2O from 10.50 to 22.73 kcal/mol) and varying degrees of conformational specificity (multistructured to singular solution structure). The singly, doubly, and triply modified (per helix) peptides can be mapped onto a contiguous segment of sequence space, providing the first experimental map of this vast molecular terrain. The energetic contours of sequence space are revealed in terms of both global folding energies (−ΔGH2O) and degree of conformational specificity within the hydrophobic core. Remarkably, six of the peptides studied (30%) contain uniquely structured hydrophobic cores amenable for NMR structural determination. The map of sequence space readily identifies a plastic site within the protein, a position which can be occupied by various amino acids with retention of a uniquely structured global fold, thereby providing a possible route for iterative redesign toward chemical enzymatic function.
Article
This report describes a joint hybridoma and combinatorial antibody library approach to elicit catalysts for primary amide bond hydrolysis. By immunization with a trigonal boronic acid hapten 3a and construction of a Fab (antigen-binding fragment) library, a diastereoselective catalyst for hydrolysis of the tripeptide primary amide substrate 1a was selected. In contrast, no antibody catalyst was isolated by standard hybridoma methods of monoclonal antibody production following immunizations with hapten 3a. The active Fab, BL25, obeys Michaelis−Menten kinetic behavior (kcat/kuncat ca. 4 × 104, Km = 150 μM) and is competitively inhibited by a boronic acid hapten analog 3b (Ki = 9 μM). Kinetic and binding studies both point to Fab selection of a hydrated tetrahedral anionic form of the boronic acid hapten 3a which serves to mimic the putative transition state 4 for catalysis of water addition to the primary amide bond. Fab-BL25 exhibits exquisite substrate selectivity, as a methyl ester analog of 1a is not accepted as a substrate. This work emphasises the power of the direct selection strategy when linked to screening of antibody combinatorial libraries and discloses the utility of boronic acids as haptens in acyl transfer processes.
Article
KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix−loop−helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive site of KO-42 contains six histidine residues with perturbed pKa values. The pKas of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s-1, which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10-5 M-1 s-1. The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M-1 s-1 which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 × 10-5 M-1 s-1. KO-42 catalyzes the corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution where the rate constant ratio k2(KO-42)/k2(4-MeIm) is larger than 103 the predominant reactive species of KO-42 have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0 which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is 1.1 which shows that there is neither general acid−general base catalysis nor strong hydrogen bonding in the transition state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that bind to one or both of the ester oxygens in the transition state.
Article
To assess the relative proficiencies of enzymes that catalyze the hydrolysis of internal and C-terminal peptide bonds, the rates of the corresponding nonenzymatic reactions were examined at elevated temperatures in sealed quartz tubes, yielding linear Arrhenius plots. The results indicate that in neutral solution at 25 °C, peptide bonds are hydrolyzed with half-times of approximately 500 years for the C-terminal bond of acetylglycylglycine, 600 years for the internal peptide bond of acetylglycylglycine N-methylamide, and 350 years for the dipeptide glycylglycine. These reactions, insensitive to changing pH or ionic strength, appear to represent uncatalyzed attack by water on the peptide bond. Comparison of rate constants indicates very strong binding of the altered substrate in the transition states for the corresponding enzyme reactions, Ktx attaining a value of less than 10-17 M in carboxypeptidase B. The half-life of the N-terminal peptide bond in glycylglycine N-methylamide, whose hydrolysis might have provided a reference for assessing the catalytic proficiency of an aminopeptidase, could not be determined because this compound undergoes relatively rapid intramolecular displacement to form diketopiperazine (t1/2 35 days at pH 7 and 37 °C). The speed of this latter process suggests an evolutionary rationale for posttranslational N-acetylation of proteins in higher organisms, as a protection against rapid degradation.
Article
In previous research, Hilvert and co-workers developed an antibody which catalyzes the decomposition of a nitrobenzisoxazole with a rate >108 times faster than the acetate-catalyzed reaction in water. Quantum mechanical calculations were carried out on a model system, the reaction of isoxazole with formate. The orientation of the carboxylate group has a significant effect on the rate. Complexation of the formate base by one water retards the reaction by approximately 5 kcal/mol; hence desolvation of the catalytic base could account for as much as four orders of magnitude in reaction rate. It was also determined that hydrogen-bonding to the forming oxide could potentially lead to greater rate acceleration. The gas phase activation barriers predict that water is the most effective general acid, lowering the activation energy by 9.5 kcal/mol. Methanol and formic acid are also effective, lowering the activation energy by 7.5 and 7.8 kcal/mol, respectively. Our calculations suggest that the combined effects of proper base orientation and acid catalysis could lead to an additional factor of 105−106 increase in rate acceleration. Based on these results, various new haptens were proposed. Each was quantitatively assessed for similarity with the located transition states to predict their potential as successful haptens.
Article
Benzisoxazoles bearing 3-hydrogens isomerize to salicylonitriles in the presence of tetrabutylammonium acetate in dipolar aprotic solvents at rates which are up to 107 times larger than those observed in water. The decarboxylation of salts of 3-carboxy-6-nitrobenzisoxazole in water is accelerated by as much as 104 by the addition of a benzonitrile phase. In striking contrast to the behavior of other 3-carboxybenzisoxazoles, the decarboxylation of 3-carboxy-4-hydroxybenzisoxazole occurs at rates which are nearly solvent independent. From these observations, it is concluded that the factors which influence the rates of decarboxylation are carboxylate ion hydrogen bond formation in protic solvents which inhibits the reaction by selectively stabilizing the starting material, and transition state stabilization in dipolar aprotic solvents which accelerates the reaction. Indirect evidence requires that carboxylate anions exist to a significant extent as nonhydrogen bonded species in benzonitrile saturated with water. The latter two factors are argued to be the essential preconditions for the construction of practical enzyme-like catalysts for this reaction.
Article
The anions of 3-carboxybenzisoxazoles undergo a quantitative decarboxylation forming salicylonitriles. The reaction is assigned an intermediateless, concerted mechanism on the basis of substituent effects and the failure to detect 33H-benzisoxazole as a product of reaction at pH 2 in the presence of tritiated water. Salt effect, pH rate profiles, and activation parameters are reported for aqueous reactions. Rate constants are reported for the tetramethylguanidine catalyzed decarboxylation of 3-carboxy-6-nitrobenzisoxazole in 24 solvents and 6 solvent mixtures and are found to vary over a range of 108. Hammett p values are reported for substituted benzisoxazoles for reaction in 14 solvents. Correlation of solvent rate data with solvent parameters of other workers is discussed.
Article
The catalytic constants for the reactions of 55 of the possible pairs of eight substituted benzisoxazoles with 12 tertiary amines in water at 30· vary over a range of 105, yet are quantitatively approximated by the equation : log kcat = 0.721pKamine + 0.614(14 -pKcyanophenol) -11.9. An examination of the above catalytic constants, together with those involving water or hydroxide ion, fails to provide evidence for a change in selectivity with reactivity for the base catalyzed E2 elimination of benzisoxazoles, despite a total variation in rate of 1011. Kinetic isotope effects for three 3-2H-substituted benzisoxazoles showed no significant variation over a range of catalytic constant values of 109. The generality and implications of these observations are discussed.
Article
The Claisen rearrangement of chorismate to prephenate and models for the catalysis of this reaction by the enzyme chorismate mutase were studied using Hartree-Fock and density functional theories. Substituent effects on the reaction are studied using several simple model systems. Whereas carboxylic acid or carboxylate substituents in the 2-position or a carboxylate in the 6-position of allyl vinyl ether leads to a lower activation energy, substitution by a carboxylic acid in the 6-position increases the activation energy. Upon 2,6-disubstitution, there is an increase in activation energy due to electrostatic repulsion between the carboxylates in the transition state. Similar results were obtained for substituted vinyl cyclohexadienyl ethers. Secondary kinetic isotope effects and substituent effects on reactant and transition state geometries are discussed. The catalysis of the reaction by the amino acid side chains in the enzyme was studied by calculation of the interaction of various functional groups that mimic the active site of chorismate mutase from Bacillus subtilis with substituted allyl vinyl ethers. Selective transition state binding by appropriately positioned hydrogen bond donors is the most important factor for catalysis, lowering the activation energy by 6 kcal/mol in the case of allyl vinyl ether-2,6-dicarboxylate. Charge complementarity to a 2-carboxylate and increased hydrogen-bonding to the ether oxygen lower the activation energy by 1.7 and 2 kcal/mol, respectively. Stabilization of the positive partial charge in the allyl part of the transition state has no significant catalytic effect. Electrophilic catalysis involving strong binding to the ether oxygen leads to a dissociative mechanism. The results are discussed with respect to the catalytic mechanism of the native enzymes and the antibody 1F7.
Article
The decarboxylation of benzisoxazole-3-carboxylate has been investigated in detail by ab initio molecular orbital calculations. The effects of solvent on transition state geometries have been investigated by inclusion of one or two water molecules in the ab initio calculations. The decarboxylation and ring opening steps are found to be concerted. Kinetic isotope effects have been calculated for the carboxylate-C-13-labeled compound for various transition state geometries. Satisfactory agreement has been found between the experimental values for the reaction in water and ab initio HF/6-31G* calculated values for systems including four hydrogen bonds to the carboxylate group. The variations in free energies of solvation along the reaction path in five different solvents (water, methanol, chloroform, acetonitrile, tetrahydrofuran) have been calculated with Monte-Carlo free energy perturbation calculations. Solvent effects are generally overestimated, but the experimental trends have been reproduced for four of the five solvents, The effects of ion pairing have been tested by inclusion of a tetramethylguanidinium cation into the Monte-Carlo simulations for acetonitrile and tetrahydrofuran. With inclusion of ion pairing, the relative rates of THF and acetonitrile are reproduced much better, but solvent effects are underestimated relative to the reaction in water.
Article
A combined Monte Carlo quantum mechanical and molecular mechanical (QM/MM) simulation method is described for the investigation of the solvent effects on chemical reactions, In the present approach, ab initio molecular orbital calculations are first used to locate the transition state, from which the reaction path is determined by using Gaussian 90. Then, free energy changes between adjacent structures generated along this intrinsic reaction path are evaluated via statistical perturbation theory using the combined QM/MM-AM1/TIP3P potential. Since empirical parametrization of the reaction system is not needed in these calculations, the method presented here is essentially an automated procedure for simulating reactions in solution, which may be conveniently used by organic chemists. We have employed the procedure to examine the decarboxylation of 3-carboxybenzisoxazole in aqueous solution. The predicted free energy of activation is 26.1 +/- 0.3 kcal/mol, in excellent agreement with the experimental value of 26.3 kcal/mol. Analyses of the contributing factors in solute-solvent interaction suggest that the aqueous solvent effect is primarily due to the difference in the intrinsic (in vacuo) charge distributions between the reactant and transition state. Solvent polarization contributes significantly to the solute-solvent interaction; however, the nature of the electronic polarization of the reactant and the transition state is markedly different.
Article
Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
Article
Die Wasserstoffbrücke ist die wichtigste der gerichteten intermolekularen Wechselwirkungen. Sie spielt für die molekulare Konformation, Aggregation und Funktion einer großen Anzahl chemischer Systeme, die vom anorganischen bis zum biologischen Sektor reichen, wichtige Rollen. Die Forschung über Wasserstoffbrücken stagnierte in den 1980er Jahren, wurde um 1990 aber wieder belebt und ist seither in schneller Bewegung. Die Wasserstoffbrücke wird heute als ein sehr breites Phänomen mit offenen Grenzen zu anderen Effekten begriffen. In den kondensierten Phasen gibt es dutzende Arten von häufig vorkommenden X−H⋅⋅⋅A-Wasserstoffbrücken und zusätzlich sehr viele weniger häufige Typen. Die Dissoziationsenergien überstreichen mehr als zwei Größenordnungen (etwa 0.2 bis 40 kcal mol−1). Innerhalb dieses Bereiches ist die Natur der Wechselwirkung nicht konstant, sondern ihre elektrostatischen, kovalenten und dispersiven Anteile variieren in ihren relativen Beiträgen. Die Wasserstoffbrücke hat breite Übergangsbereiche zur kovalenten Bindung, zur Van-der-Waals-Wechselwirkung, zur ionischen Wechselwirkung und zur Kation-π-Wechselwirkung. Wasserstoffbrücken können als beginnende Protonentransferreaktion aufgefasst werden, und für starke Wasserstoffbrücken kann diese Reaktion bereits in einem fortgeschrittenen Stadium sein. In diesem Aufsatz wird versucht, einen zusammenhängenden Überblick über alle diese Aspekte zu geben.
Article
Gleichzeitige Sättigungsmutagenese dreier Aminosäurereste der Esterase BS2 aus Bacillus subtilis und folgendes Hochdurchsatz-Screening führten zur Identifizierung einer Doppelmutante (E188W/M193C). Diese zeigt, anders als die Einzelmutanten, eine umgekehrte Enantiopräferenz wie der Wildtyp (WT) (siehe Schema für die Racematspaltung des Esters 1) sowie einen hohen E-Wert und ein erweitertes Substratspektrum.
Article
Modern tools for enzyme discovery and protein engineering substantially broadened the number of enzymes applicable for biocatalysis and helped to alter their properties such as substrate range, enantioselectivity, and stability under process conditions. In addition, these methods also enabled one to explore reactions for organic synthesis for which no suitable enzymes were available until recently. This review provides a summary of the different concepts and technologies, which are exemplified for various enzymes.
Article
Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.