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Thomas R Ward - One of the best experts on this subject based on the ideXlab platform.
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directed evolution of artificial Metalloenzymes for in vivo metathesis
Nature, 2016Co-Authors: Markus Jeschek, Raphael Reuter, Christian Trindler, Juliane Klehr, Tillmann Heinisch, Sven Panke, Thomas R WardAbstract:An artificial Metalloenzyme is compartmentalized and evolved in vivo for olefin metathesis—an archetypal organometallic reaction without equivalent in nature; the evolved metathase reveals broad substrate scope and compares favourably with commercial catalysts. Artificial Metalloenzymes—made by incorporating an abiotic cofactor within a protein scaffold—have the potential to engineer non-natural in vivo reactions. To be of practical use, such catalysts must maintain their activity in a cellular environment, which means overcoming the tendency of introduced metal cofactors to be inhibited by cellular components. This paper demonstrates that directed evolution can overcome this difficulty. Markus Jeschek et al. report on the in vivo evolution of a ruthenium–protein complex that can catalyse olefin metathesis—an archetypal organometallic reaction with no equivalent in nature—in the periplasm of Escherichia coli. The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the protocol. The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. For example, artificial Metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist3: repurposing natural Metalloenzymes for abiotic transformations2,4; in silico Metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. The third strategy offers the opportunity to design a wide variety of artificial Metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein12,13,14,15. This limits the throughput of genetic optimization schemes applied to artificial Metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial Metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial Metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor15. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial Metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.
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Directed evolution of artificial Metalloenzymes for in vivo metathesis
Nature, 2016Co-Authors: Markus Jeschek, Raphael Reuter, Christian Trindler, Juliane Klehr, Tillmann Heinisch, Sven Panke, Thomas R WardAbstract:The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial Metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural Metalloenzymes for abiotic transformations; in silico Metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial Metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial Metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial Metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the 'metathase' because it offers an auspicious environment for artificial Metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial Metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.
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genetic optimization of Metalloenzymes enhancing enzymes for non natural reactions
Angewandte Chemie, 2016Co-Authors: Todd K Hyster, Thomas R WardAbstract:Artificial Metalloenzymes have received increasing attention over the last decade as a possible solution to unaddressed challenges in synthetic organic chemistry. Whereas traditional transition-metal catalysts typically only take advantage of the first coordination sphere to control reactivity and selectivity, artificial Metalloenzymes can modulate both the first and second coordination spheres. This difference can manifest itself in reactivity profiles that can be truly unique to artificial Metalloenzymes. This Review summarizes attempts to modulate the second coordination sphere of artificial Metalloenzymes by using genetic modifications of the protein sequence. In doing so, successful attempts and creative solutions to address the challenges encountered are highlighted.
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Latest Developments in Metalloenzyme Design and Repurposing
European Journal of Inorganic Chemistry, 2015Co-Authors: Tillmann Heinisch, Thomas R WardAbstract:In the past decade, artificial Metalloenzymes (AMEs) have emerged as attractive alternatives to more traditional homogeneous catalysts and enzymes. This microreview presents a selection of recent achievements in the design of such hybrid catalysts. These include artificial zinc hydrolases and metathesases, the heme-protein repurposing for C–H, N–H, and S–H insertion reactions, novel light-driven redox hybrid catalysts, novel scaffold proteins, and metallocofactor anchoring techniques and Metalloenzyme models.
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recent achievments in the design and engineering of artificial Metalloenzymes
Current Opinion in Chemical Biology, 2014Co-Authors: Marc Durrenberger, Thomas R WardAbstract:Herein, we highlight a selection of recent successes in the creation of artificial Metalloenzymes. A particular emphasis is set on different anchoring methods to incorporate the abiotic metal cofactor within the host protein as well as promising strategies for the de novo design of artificial Metalloenzymes. Both approaches yield promiscuous catalytic activities which expand the catalytic repertoire of biocatalysis and synthetic biology. Moreover, we summarize laboratory evolution protocols which have contributed to unravel the full potential of artificial Metalloenzymes.
Seth M Cohen - One of the best experts on this subject based on the ideXlab platform.
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targeting Metalloenzymes for therapeutic intervention
Chemical Reviews, 2019Co-Authors: Allie Y Chen, Rebecca N. Adamek, Cy V. Credille, Benjamin L. Dick, Christine N Morrison, Seth M CohenAbstract:Metalloenzymes are central to a wide range of essential biological activities, including nucleic acid modification, protein degradation, and many others. The role of Metalloenzymes in these processes also makes them central for the progression of many diseases and, as such, makes Metalloenzymes attractive targets for therapeutic intervention. Increasing awareness of the role Metalloenzymes play in disease and their importance as a class of targets has amplified interest in the development of new strategies to develop inhibitors and ultimately useful drugs. In this Review, we provide a broad overview of several drug discovery efforts focused on Metalloenzymes and attempt to map out the current landscape of high-value Metalloenzyme targets.
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metal binding isosteres as new scaffolds for Metalloenzyme inhibitors
Inorganic Chemistry, 2018Co-Authors: Benjamin L. Dick, Seth M CohenAbstract:The principle of isosteres or bioisosteres in medicinal chemistry is a central and essential concept in modern drug discovery. For example, carboxylic acids are often replaced by bioisosteres to mitigate issues related to lipophilicity or acidity while retaining acidic characteristics in addition to hydrogen bond donor/acceptor abilities. Separately, the development of metal-binding pharmacophores (MBPs) for binding to the active site metal ion in Metalloenzymes of therapeutic interest is an emerging area in the realm of fragment-based drug discovery (FBDD). The direct application of the bioisostere concept to MBPs has not been well-described or systematically investigated. Herein, the picolinic acid MBP is used as a case study for the development of MBP isosteres (so-called MBIs). Many of these isosteres are novel compounds, and data on their physicochemical properties, metal binding capacity, and Metalloenzyme inhibition characteristics are presented. The results show that MBIs of picolinic acid generally retain metal coordinating properties and exhibit predictable Metalloenzyme inhibitory activity while possessing a broad range of physicochemical properties (e.g., p Ka, log P). These findings demonstrate the use of bioisosteres results in an untapped source of metal binding functional groups suitable for Metalloenzyme FBDD. These MBIs provide a previously unexplored route for modulating the physicochemical properties of Metalloenzyme inhibitors and improving their drug-likeness.
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Isosteres of hydroxypyridinethione as drug-like pharmacophores for Metalloenzyme inhibition.
Journal of Biological Inorganic Chemistry, 2018Co-Authors: Rebecca N. Adamek, Cy V. Credille, Benjamin L. Dick, Seth M CohenAbstract:Hydroxypyridinethiones (HOPTOs) are strong ligands for metal ions and potentially useful pharmacophores for inhibiting Metalloenzymes relevant to human disease. However, HOPTOs have been sparingly used in drug discovery efforts due, in part, to concerns that this scaffold will act as a promiscuous, non-selective Metalloenzyme inhibitor, as well as possess poor pharmacokinetics (PK), which may undermine drug candidates containing this functional group. To advance HOPTOs as a useful pharmacophore for Metalloenzyme inhibitors, a library of 22 HOPTO isostere compounds has been synthesized and investigated. This library demonstrates that it is possible to maintain the core metal-binding pharmacophore (MBP) while generating diversity in structure, electronics, and PK properties. This HOPTO library has been screened against a set of four different Metalloenzymes, demonstrating that while the same metal-binding donor atoms are maintained, there is a wide range of activity between Metalloenzyme targets. Overall, this work shows that HOPTO isosteres are useful MBPs and valuable scaffolds for Metalloenzyme inhibitors.
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Metal-Binding Isosteres as New Scaffolds for Metalloenzyme Inhibitors
2018Co-Authors: Benjamin L. Dick, Seth M CohenAbstract:The principle of isosteres or bioisosteres in medicinal chemistry is a central and essential concept in modern drug discovery. For example, carboxylic acids are often replaced by bioisosteres to mitigate issues related to lipophilicity or acidity while retaining acidic characteristics in addition to hydrogen bond donor/acceptor abilities. Separately, the development of metal-binding pharmacophores (MBPs) for binding to the active site metal ion in Metalloenzymes of therapeutic interest is an emerging area in the realm of fragment-based drug discovery (FBDD). The direct application of the bioisostere concept to MBPs has not been well-described or systematically investigated. Herein, the picolinic acid MBP is used as a case study for the development of MBP isosteres (so-called MBIs). Many of these isosteres are novel compounds, and data on their physicochemical properties, metal binding capacity, and Metalloenzyme inhibition characteristics are presented. The results show that MBIs of picolinic acid generally retain metal coordinating properties and exhibit predictable Metalloenzyme inhibitory activity while possessing a broad range of physicochemical properties (e.g., pKa, logP). These findings demonstrate the use of bioisosteres results in an untapped source of metal binding functional groups suitable for Metalloenzyme FBDD. These MBIs provide a previously unexplored route for modulating the physicochemical properties of Metalloenzyme inhibitors and improving their drug-likeness
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a bioinorganic approach to fragment based drug discovery targeting Metalloenzymes
Accounts of Chemical Research, 2017Co-Authors: Seth M CohenAbstract:ConspectusMetal-dependent enzymes (i.e., Metalloenzymes) make up a large fraction of all enzymes and are critically important in a wide range of biological processes, including DNA modification, protein homeostasis, antibiotic resistance, and many others. Consequently, Metalloenzymes represent a vast and largely untapped space for drug development. The discovery of effective therapeutics that target Metalloenzymes lies squarely at the interface of bioinorganic and medicinal chemistry and requires expertise, methods, and strategies from both fields to mount an effective campaign. In this Account, our research program that brings together the principles and methods of bioinorganic and medicinal chemistry are described, in an effort to bridge the gap between these fields and address an important class of medicinal targets.Fragment-based drug discovery (FBDD) is an important drug discovery approach that is particularly well suited for Metalloenzyme inhibitor development. FBDD uses relatively small but diverse...
Gerard Roelfes - One of the best experts on this subject based on the ideXlab platform.
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supramolecular assembly of artificial Metalloenzymes based on the dimeric protein lmrr as promiscuous scaffold
Journal of the American Chemical Society, 2015Co-Authors: Wesley R. Browne, Arnold J. M. Driessen, Gerard RoelfesAbstract:Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial Metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial Metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel–Crafts alkylation of indoles.
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artificial Metalloenzymes for asymmetric catalysis by creation of novel active sites in protein and dna scaffolds
Israel Journal of Chemistry, 2015Co-Authors: Ivana Drienovska, Gerard RoelfesAbstract:Artificial Metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial Metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial Metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial Metalloenzymes.
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novel artificial Metalloenzymes by in vivo incorporation of metal binding unnatural amino acids
Chemical Science, 2015Co-Authors: Ivana Drienovska, Ana Riozmartinez, Apparao Draksharapu, Gerard RoelfesAbstract:Artificial Metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial Metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial Metalloenzymes were applied in catalytic asymmetric Friedel–Crafts alkylation reactions and up to 83% ee for the product was achieved.
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artificial Metalloenzymes for enantioselective catalysis
Current Opinion in Chemical Biology, 2014Co-Authors: Gerard RoelfesAbstract:Artificial Metalloenzymes have emerged over the last decades as an attractive approach towards combining homogeneous catalysis and biocatalysis. A wide variety of catalytic transformations have been established by artificial Metalloenzymes, thus establishing proof of concept. The field is now slowly transforming to take on new challenges. These include novel designs, novel catalytic reactions, some of which have no equivalent in both homogenous catalysis and biocatalysis and the incorporation of artificial Metalloenzymes in chemoenzymatic cascades. Some of these developments represent promising steps towards integrating artificial Metalloenzymes in biological systems. This review will focus on advances in this field and perspectives discussed.
Mingpan Cheng - One of the best experts on this subject based on the ideXlab platform.
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enantioselective sulfoxidation reaction catalyzed by a g quadruplex dna Metalloenzyme
Chemical Communications, 2016Co-Authors: Mingpan Cheng, Jun Zhou, Guoqing Jia, Yan YangAbstract:Enantioselective sulfoxidation reaction is achieved for the first time by a DNA Metalloenzyme assembled with the human telomeric G-quadruplex DNA and Cu(II)–4,4′-bimethyl-2,2′-bipyridine complex, and the mixed G-quadruplex architectures are responsible for the catalytic enantioselectivity and activity.
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terpyridine cu ii targeting human telomeric dna to produce highly stereospecific g quadruplex dna Metalloenzyme
Chemical Science, 2015Co-Authors: Mingpan Cheng, Jingya Hao, Changhao Wang, Guoqing JiaAbstract:The cofactors commonly involved in natural enzymes have provided the inspiration for numerous advances in the creation of artificial Metalloenzymes. Nevertheless, to design an appropriate cofactor for a given biomolecular scaffold or vice versa remains a challenge in developing efficient catalysts in biochemistry. Herein, we extend the idea of G-quadruplex-targeting anticancer drug design to construct a G-quadruplex DNA Metalloenzyme. We found that a series of terpyridine–Cu(II) complexes (CuLn) can serve as excellent cofactors to dock with human telemetric G-quadruplex DNA. The resulting G-quadruplex DNA Metalloenzyme utilising CuL1 catalyzes an enantioselective Diels–Alder reaction with enantioselectivity of >99% enantiomeric excess and about 73-fold rate acceleration compared to CuL1 alone. The terpyridine–Cu(II) complex cofactors demonstrate dual functions, both as an active site to perform catalysis and as a structural regulator to promote the folding of human telemetric G-quadruplex DNA towards excellent catalysts.
Tillmann Heinisch - One of the best experts on this subject based on the ideXlab platform.
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directed evolution of artificial Metalloenzymes for in vivo metathesis
Nature, 2016Co-Authors: Markus Jeschek, Raphael Reuter, Christian Trindler, Juliane Klehr, Tillmann Heinisch, Sven Panke, Thomas R WardAbstract:An artificial Metalloenzyme is compartmentalized and evolved in vivo for olefin metathesis—an archetypal organometallic reaction without equivalent in nature; the evolved metathase reveals broad substrate scope and compares favourably with commercial catalysts. Artificial Metalloenzymes—made by incorporating an abiotic cofactor within a protein scaffold—have the potential to engineer non-natural in vivo reactions. To be of practical use, such catalysts must maintain their activity in a cellular environment, which means overcoming the tendency of introduced metal cofactors to be inhibited by cellular components. This paper demonstrates that directed evolution can overcome this difficulty. Markus Jeschek et al. report on the in vivo evolution of a ruthenium–protein complex that can catalyse olefin metathesis—an archetypal organometallic reaction with no equivalent in nature—in the periplasm of Escherichia coli. The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the protocol. The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. For example, artificial Metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist3: repurposing natural Metalloenzymes for abiotic transformations2,4; in silico Metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. The third strategy offers the opportunity to design a wide variety of artificial Metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein12,13,14,15. This limits the throughput of genetic optimization schemes applied to artificial Metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial Metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial Metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor15. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial Metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.
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Directed evolution of artificial Metalloenzymes for in vivo metathesis
Nature, 2016Co-Authors: Markus Jeschek, Raphael Reuter, Christian Trindler, Juliane Klehr, Tillmann Heinisch, Sven Panke, Thomas R WardAbstract:The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial Metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural Metalloenzymes for abiotic transformations; in silico Metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial Metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial Metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial Metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the 'metathase' because it offers an auspicious environment for artificial Metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial Metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.
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Latest Developments in Metalloenzyme Design and Repurposing
European Journal of Inorganic Chemistry, 2015Co-Authors: Tillmann Heinisch, Thomas R WardAbstract:In the past decade, artificial Metalloenzymes (AMEs) have emerged as attractive alternatives to more traditional homogeneous catalysts and enzymes. This microreview presents a selection of recent achievements in the design of such hybrid catalysts. These include artificial zinc hydrolases and metathesases, the heme-protein repurposing for C–H, N–H, and S–H insertion reactions, novel light-driven redox hybrid catalysts, novel scaffold proteins, and metallocofactor anchoring techniques and Metalloenzyme models.
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a dual anchoring strategy for the localization and activation of artificial Metalloenzymes based on the biotin streptavidin technology
Journal of the American Chemical Society, 2013Co-Authors: Jeremy Malcolm Zimbron, Tillmann Heinisch, Maurus Schmid, Didier R Hamels, Elisa S Nogueira, Tilman Schirmer, Thomas R WardAbstract:Artificial Metalloenzymes result from anchoring an active catalyst within a protein environment. Toward this goal, various localization strategies have been pursued: covalent, supramolecular, or dative anchoring. Herein we show that introduction of a suitably positioned histidine residue contributes to firmly anchor, via a dative bond, a biotinylated rhodium piano stool complex within streptavidin. The in silico design of the artificial Metalloenzyme was confirmed by X-ray crystallography. The resulting artificial Metalloenzyme displays significantly improved catalytic performance, both in terms of activity and selectivity in the transfer hydrogenation of imines. Depending on the position of the histidine residue, both enantiomers of the salsolidine product can be obtained.