Prof. Dr. Thomas R. Ward Department of Chemistry Profiles & Affiliations OverviewResearch Publications Projects & Collaborations Projects & Collaborations OverviewResearch Publications Projects & Collaborations Profiles & Affiliations Projects & Collaborations 22 foundShow per page10 10 20 50 MetRaZymes - Metal-containing Radical Enzymes Research Project | 1 Project MembersNo Description available NCCR Catalysis - Sustainable Chemical Processes Through Catalysis Research Project | 1 Project MembersSustainable chemical processes through catalyst design. NCCR Molecular Systems Engineering - phase 2 Research Project | 30 Project MembersMolecular Systems Engineering is a National Centre of Competence in Research (NCCR) funded by the Swiss National Science Foundation (SNSF), and headed by the University of Basel and the ETH Zurich. This NCCR combines expertise from chemistry, biology, physics, bioinformatics, and engineering. The overreaching aim is to develop tools and devices to monitor and manipulate off-equilibrium (bio)chemical systems. These may find applications in the synthesis of high added-value products, as innovative diagnostic tools and for the restoration of a desired cellular or organ function. EMBO ALTF 194-2017, Long-Term Fellowship Research Project | 2 Project MembersThe emerging field of synthetic biology offers new opportunities to address current biological and medical challenges, by providing tools to label and influence metabolic pathways as well as approaches for the development of novel therapeutic strategies. Particularly, a catalytic approach based on Artificial Metalloenzymes (AMs) could play a fundamental role in this endeavor. This project outlines a strategy for the development of a new tumor treatment therapy. To this end, the cell surface tumor marker carbonic anhydrase IX (CAIX) is 'hijacked' as an AM by binding a CA-inhibitor, bearing a metallocofactor, to the Zn(II) ion in the active site of CA. Upon binding, the metallocofactor is activated and catalyzes the uncaging of a prodrug thus releasing a potent chemotherapeutic drug. As a complementary approach, I seek to improve the performance of CA-based AMs in cellulo by combining protein design with in vivo evolution. AMs are designed and evolved to complement existing metabolic pathways. Specifically, to form the valine precursor α-ketovaline in an auxotrophic Escherichia coli knockout strain. This project is a proof of principle and blueprint for the development of AM-based tools for studying cellular regulation to answer basic biological questions. Interactions, dynamics and functionality at nanoscale characterized by confocal laser scanning microscopy and fluorescence correlation spectroscopy Research Project | 5 Project MembersFluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and Förster resonance energy transfer (FRET) play an irreplaceable role to determine the number and movement of molecules, biological entities, and nanoparticles in biological or synthetic systems. These methods can analyze particles, not only in solution, but also attached to surfaces, inside cells, or nanoparticles. From these measurements, the number of particles, binding strength (KD), distance, surface modifications, and the speed of particles can be determined. When coupled with confocal laser scanning microscopy (CLSM), the particles can be visualized in various environments as well. We require financial support for a new state of the art CLSM/FCS/FCCS instrument in the Department of Chemistry at the University of Basel that will include all three spectroscopy modes (FCS, FCCS and FRET) as well as a high resolution confocal microscope and cell incubation platform. We strongly depend on these techniques in our on-going and future projects. This instrument will allow for resolving images of fluorescently labeled particles from the nano to micrometer size (~140 nm- 2 µm), as well as enable the long term study of particles inside growing cells. FCS and FCCS will allow for measurements of product formation and molecule localization at the site of interest, as well as allow for the determination of binding, release, and movement of particles. It will also expand our capabilities by giving us the ability to detect samples inside growing cells for extended periods of time, which was previously not possible. The new software will make data analysis and interpretation much simpler, and with models adapted for complex systems. Finally, the system is designed in a modular fashion allowing for the future upgrade with more diverse laser arrays as well as the addition of enhanced fluorescence lifetime imaging microscopy. This new state of the art CLSM/FCS/FCCS instrument will support research emphasized by the University of Basel, within the scope of "life- and nanoscience. It will ensure the international competitiveness of the research groups involved in the proposal (groups from the Department of Chemistry, Biozentrum, and the Swiss Tropical and Public Health Institute), and their collaborations within the NCCR Molecular Systems Engineering, the Swiss Nanoscience Institute, and industrial partners. Moreover, other groups from adjacent departments of the University of Basel and various industrial partners can have access based on joint projects. Picosecond Transient Absorption Setup for Detection of Short-Lived Photoproducts and Excited States in Molecular Systems Research Project | 6 Project MembersMany photophysical and photochemical processes which are relevant for light-to-chemical energy conversion occur on very rapid timescales. Time-resolved UV-Vis absorption spectroscopy has become an indispensable tool in modern photochemistry. Several ongoing Ph. D. theses and postdoctoral research projects in the main applicant's group ask for a transient absorption spectrometer with picosecond time resolution and an appropriate laser source. Among these projects are for example the investigation of photoinduced multi-electron transfer reactions in order to spatially separate multiple electrons from multiple holes, which is of key importance for producing chemical fuels with sunlight as energy input (projects 1 and 2). Similarly, picosecond transient absorption spectrosocopy will permit mechanistic studies of photoinduced proton-coupled electron transfer (PCET) reactions which will greatly further our current fundamental understanding of this important class of reactions (project 3). The activation of small inert molecules such as H 2 O, CO 2 or N 2 will invariably rely on multi-electron, multi-proton chemistry hence the proposed photochemical studies are important in the greater context of solar energy conversion. NCCR Molecular Systems Engineering Research Project | 33 Project MembersThe NCCR Molecular Systems Engineering combines competences from life sciences, chemistry, physics, biology, bioinformatics and engineering sciences. More than 100 researchers and support personnel distributed into four work packages and 31 projects work together to address systems engineering challenges by integrating novel chemical and biological modules into molecular factories and cellular systems for the production of high added-value chemicals and applications in medical diagnostics and treatment. Molecular systems engineering relies on the combination of both chemical- and biological modules. In this approach, complex dynamic phenomena emerge as the result of the integration of molecular modules designed to interact in a programmed way with their complex environment. In this manner, it should be possible to create molecular factories and cellular systems whose properties are more than the sum of the attributes of the individual modules. The commitments of the leading houses, the University of Basel and ETH Zurich, also include new (joint) professorships, and extensive training of a new generation of scientists and technologists, leading to a long-term paradigm shift in molecular sciences and a new structure of the Swiss research landscape. NCCR MSE - Molecular Factories Based on Artificial Metalloenzymes Research Project | 3 Project MembersArtificial metalloenzymes (ArMs) result from the incorporation of abiotic cofactors within a host protein. In collaboration with various groups of the NCCR, we aim at engineering metabolic pathways incorporating ArMs. The challenges addressed during this funding period include: i) successful compartmentalization and evolution of ArMs within the periplasm of E coli, ii) engineering enzyme cascades with ArMs and iii) cross-regulation of ArMs. Artificial Metalloenzymes: Supramolecular Anchoring and Activation Research Project | 1 Project MembersLife emerges from inanimate matter thanks to a complex network of tightly cross-regulated enzymatic reaction cascades. To mimick this, we have emplemented the cross-regulation of an artificial imine reductase which is turned on in the presence of a protease, generating a tripeptide that acts as a cross-regulator for the imine reductase. Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications Research Project | 1 Project MembersArtificial metalloenzymes result from combining a catalytically competent organometallic moiety with a host protein. The resulting hybrid catalyst combine attractive features of both chemo- and biocatalysts. In recent years, the Ward group has exploited the biotin-streptavidin towards the creation of artificial metalloenzymes for hydrogenation, allylic alkylation, sulfoxidation, alcohol oxidation, dihydroxylation, transfer-hydrogenation and olefin metathesis. The latter two systems were shown to be particularly stable towards E. coli cellular extracts. Within this funding period, it is proposed to exploit this finding towards the implementation of directed evolution protocols for the optimization of the performance of artificial metalloenzymes. Four complementary and intedisciplinary sub-projects will be investigated: i) exploiting streptavidin expressed in the periplasm; ii) cascade reactions with artificial metalloenzymes; iii) optimization of artificial transfer-hydrogenase for the production of high-added value amines and aminoacids and iv) directed evolution of artificial metathesases. i) In order to circumvent the inhibition of the biotinylated precious metal catalyst by glutathione (present in milimolar amounts in the cytoplasm), it is proposed to target streptavidin to the periplasm. This will allow us to sidestep the lengthy purification of streptavidin prior to catalysis, eventually allowing the implementation of directed evolution protocols. ii) We have shown that artificial metalloenzymes are compatible with a variety of biocatalysts. Combining artificial metalloenyzmes with natural enzymes will lead to complex reaction cascades that can be used a) as a high-throughput colorimetric assay or b) to complement metabolic pathways. iii) The above developments will be exploited towards the preparation of a) high-added value amines via the enantioselective imine reduction and b) leucine by relying on a selection strategy based on E. coli leucine auxotrophs. iv) Thanks to the inertness of artificial metathesases based on the biotin-streptavidin technology, the performance of these will be optmized using crude E. coli cell extracts. For this purpose, we will rely screening a fluorophore-quencher substrate which, upon ring closing metathesis releases the quencher, thus becoming fluorescent. Ultimately, we aim at developing artificial metalloenzymes that outperform classical organometallic catalysts. In a biomimetic spirit and thanks to Darwinian protocols, we anticipate that the presence of an optimized second coordination sphere provided by the protein environment will allow to achieve this ambitious goal. 123 123 OverviewResearch Publications Projects & Collaborations
Projects & Collaborations 22 foundShow per page10 10 20 50 MetRaZymes - Metal-containing Radical Enzymes Research Project | 1 Project MembersNo Description available NCCR Catalysis - Sustainable Chemical Processes Through Catalysis Research Project | 1 Project MembersSustainable chemical processes through catalyst design. NCCR Molecular Systems Engineering - phase 2 Research Project | 30 Project MembersMolecular Systems Engineering is a National Centre of Competence in Research (NCCR) funded by the Swiss National Science Foundation (SNSF), and headed by the University of Basel and the ETH Zurich. This NCCR combines expertise from chemistry, biology, physics, bioinformatics, and engineering. The overreaching aim is to develop tools and devices to monitor and manipulate off-equilibrium (bio)chemical systems. These may find applications in the synthesis of high added-value products, as innovative diagnostic tools and for the restoration of a desired cellular or organ function. EMBO ALTF 194-2017, Long-Term Fellowship Research Project | 2 Project MembersThe emerging field of synthetic biology offers new opportunities to address current biological and medical challenges, by providing tools to label and influence metabolic pathways as well as approaches for the development of novel therapeutic strategies. Particularly, a catalytic approach based on Artificial Metalloenzymes (AMs) could play a fundamental role in this endeavor. This project outlines a strategy for the development of a new tumor treatment therapy. To this end, the cell surface tumor marker carbonic anhydrase IX (CAIX) is 'hijacked' as an AM by binding a CA-inhibitor, bearing a metallocofactor, to the Zn(II) ion in the active site of CA. Upon binding, the metallocofactor is activated and catalyzes the uncaging of a prodrug thus releasing a potent chemotherapeutic drug. As a complementary approach, I seek to improve the performance of CA-based AMs in cellulo by combining protein design with in vivo evolution. AMs are designed and evolved to complement existing metabolic pathways. Specifically, to form the valine precursor α-ketovaline in an auxotrophic Escherichia coli knockout strain. This project is a proof of principle and blueprint for the development of AM-based tools for studying cellular regulation to answer basic biological questions. Interactions, dynamics and functionality at nanoscale characterized by confocal laser scanning microscopy and fluorescence correlation spectroscopy Research Project | 5 Project MembersFluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and Förster resonance energy transfer (FRET) play an irreplaceable role to determine the number and movement of molecules, biological entities, and nanoparticles in biological or synthetic systems. These methods can analyze particles, not only in solution, but also attached to surfaces, inside cells, or nanoparticles. From these measurements, the number of particles, binding strength (KD), distance, surface modifications, and the speed of particles can be determined. When coupled with confocal laser scanning microscopy (CLSM), the particles can be visualized in various environments as well. We require financial support for a new state of the art CLSM/FCS/FCCS instrument in the Department of Chemistry at the University of Basel that will include all three spectroscopy modes (FCS, FCCS and FRET) as well as a high resolution confocal microscope and cell incubation platform. We strongly depend on these techniques in our on-going and future projects. This instrument will allow for resolving images of fluorescently labeled particles from the nano to micrometer size (~140 nm- 2 µm), as well as enable the long term study of particles inside growing cells. FCS and FCCS will allow for measurements of product formation and molecule localization at the site of interest, as well as allow for the determination of binding, release, and movement of particles. It will also expand our capabilities by giving us the ability to detect samples inside growing cells for extended periods of time, which was previously not possible. The new software will make data analysis and interpretation much simpler, and with models adapted for complex systems. Finally, the system is designed in a modular fashion allowing for the future upgrade with more diverse laser arrays as well as the addition of enhanced fluorescence lifetime imaging microscopy. This new state of the art CLSM/FCS/FCCS instrument will support research emphasized by the University of Basel, within the scope of "life- and nanoscience. It will ensure the international competitiveness of the research groups involved in the proposal (groups from the Department of Chemistry, Biozentrum, and the Swiss Tropical and Public Health Institute), and their collaborations within the NCCR Molecular Systems Engineering, the Swiss Nanoscience Institute, and industrial partners. Moreover, other groups from adjacent departments of the University of Basel and various industrial partners can have access based on joint projects. Picosecond Transient Absorption Setup for Detection of Short-Lived Photoproducts and Excited States in Molecular Systems Research Project | 6 Project MembersMany photophysical and photochemical processes which are relevant for light-to-chemical energy conversion occur on very rapid timescales. Time-resolved UV-Vis absorption spectroscopy has become an indispensable tool in modern photochemistry. Several ongoing Ph. D. theses and postdoctoral research projects in the main applicant's group ask for a transient absorption spectrometer with picosecond time resolution and an appropriate laser source. Among these projects are for example the investigation of photoinduced multi-electron transfer reactions in order to spatially separate multiple electrons from multiple holes, which is of key importance for producing chemical fuels with sunlight as energy input (projects 1 and 2). Similarly, picosecond transient absorption spectrosocopy will permit mechanistic studies of photoinduced proton-coupled electron transfer (PCET) reactions which will greatly further our current fundamental understanding of this important class of reactions (project 3). The activation of small inert molecules such as H 2 O, CO 2 or N 2 will invariably rely on multi-electron, multi-proton chemistry hence the proposed photochemical studies are important in the greater context of solar energy conversion. NCCR Molecular Systems Engineering Research Project | 33 Project MembersThe NCCR Molecular Systems Engineering combines competences from life sciences, chemistry, physics, biology, bioinformatics and engineering sciences. More than 100 researchers and support personnel distributed into four work packages and 31 projects work together to address systems engineering challenges by integrating novel chemical and biological modules into molecular factories and cellular systems for the production of high added-value chemicals and applications in medical diagnostics and treatment. Molecular systems engineering relies on the combination of both chemical- and biological modules. In this approach, complex dynamic phenomena emerge as the result of the integration of molecular modules designed to interact in a programmed way with their complex environment. In this manner, it should be possible to create molecular factories and cellular systems whose properties are more than the sum of the attributes of the individual modules. The commitments of the leading houses, the University of Basel and ETH Zurich, also include new (joint) professorships, and extensive training of a new generation of scientists and technologists, leading to a long-term paradigm shift in molecular sciences and a new structure of the Swiss research landscape. NCCR MSE - Molecular Factories Based on Artificial Metalloenzymes Research Project | 3 Project MembersArtificial metalloenzymes (ArMs) result from the incorporation of abiotic cofactors within a host protein. In collaboration with various groups of the NCCR, we aim at engineering metabolic pathways incorporating ArMs. The challenges addressed during this funding period include: i) successful compartmentalization and evolution of ArMs within the periplasm of E coli, ii) engineering enzyme cascades with ArMs and iii) cross-regulation of ArMs. Artificial Metalloenzymes: Supramolecular Anchoring and Activation Research Project | 1 Project MembersLife emerges from inanimate matter thanks to a complex network of tightly cross-regulated enzymatic reaction cascades. To mimick this, we have emplemented the cross-regulation of an artificial imine reductase which is turned on in the presence of a protease, generating a tripeptide that acts as a cross-regulator for the imine reductase. Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications Research Project | 1 Project MembersArtificial metalloenzymes result from combining a catalytically competent organometallic moiety with a host protein. The resulting hybrid catalyst combine attractive features of both chemo- and biocatalysts. In recent years, the Ward group has exploited the biotin-streptavidin towards the creation of artificial metalloenzymes for hydrogenation, allylic alkylation, sulfoxidation, alcohol oxidation, dihydroxylation, transfer-hydrogenation and olefin metathesis. The latter two systems were shown to be particularly stable towards E. coli cellular extracts. Within this funding period, it is proposed to exploit this finding towards the implementation of directed evolution protocols for the optimization of the performance of artificial metalloenzymes. Four complementary and intedisciplinary sub-projects will be investigated: i) exploiting streptavidin expressed in the periplasm; ii) cascade reactions with artificial metalloenzymes; iii) optimization of artificial transfer-hydrogenase for the production of high-added value amines and aminoacids and iv) directed evolution of artificial metathesases. i) In order to circumvent the inhibition of the biotinylated precious metal catalyst by glutathione (present in milimolar amounts in the cytoplasm), it is proposed to target streptavidin to the periplasm. This will allow us to sidestep the lengthy purification of streptavidin prior to catalysis, eventually allowing the implementation of directed evolution protocols. ii) We have shown that artificial metalloenzymes are compatible with a variety of biocatalysts. Combining artificial metalloenyzmes with natural enzymes will lead to complex reaction cascades that can be used a) as a high-throughput colorimetric assay or b) to complement metabolic pathways. iii) The above developments will be exploited towards the preparation of a) high-added value amines via the enantioselective imine reduction and b) leucine by relying on a selection strategy based on E. coli leucine auxotrophs. iv) Thanks to the inertness of artificial metathesases based on the biotin-streptavidin technology, the performance of these will be optmized using crude E. coli cell extracts. For this purpose, we will rely screening a fluorophore-quencher substrate which, upon ring closing metathesis releases the quencher, thus becoming fluorescent. Ultimately, we aim at developing artificial metalloenzymes that outperform classical organometallic catalysts. In a biomimetic spirit and thanks to Darwinian protocols, we anticipate that the presence of an optimized second coordination sphere provided by the protein environment will allow to achieve this ambitious goal. 123 123
MetRaZymes - Metal-containing Radical Enzymes Research Project | 1 Project MembersNo Description available
NCCR Catalysis - Sustainable Chemical Processes Through Catalysis Research Project | 1 Project MembersSustainable chemical processes through catalyst design.
NCCR Molecular Systems Engineering - phase 2 Research Project | 30 Project MembersMolecular Systems Engineering is a National Centre of Competence in Research (NCCR) funded by the Swiss National Science Foundation (SNSF), and headed by the University of Basel and the ETH Zurich. This NCCR combines expertise from chemistry, biology, physics, bioinformatics, and engineering. The overreaching aim is to develop tools and devices to monitor and manipulate off-equilibrium (bio)chemical systems. These may find applications in the synthesis of high added-value products, as innovative diagnostic tools and for the restoration of a desired cellular or organ function.
EMBO ALTF 194-2017, Long-Term Fellowship Research Project | 2 Project MembersThe emerging field of synthetic biology offers new opportunities to address current biological and medical challenges, by providing tools to label and influence metabolic pathways as well as approaches for the development of novel therapeutic strategies. Particularly, a catalytic approach based on Artificial Metalloenzymes (AMs) could play a fundamental role in this endeavor. This project outlines a strategy for the development of a new tumor treatment therapy. To this end, the cell surface tumor marker carbonic anhydrase IX (CAIX) is 'hijacked' as an AM by binding a CA-inhibitor, bearing a metallocofactor, to the Zn(II) ion in the active site of CA. Upon binding, the metallocofactor is activated and catalyzes the uncaging of a prodrug thus releasing a potent chemotherapeutic drug. As a complementary approach, I seek to improve the performance of CA-based AMs in cellulo by combining protein design with in vivo evolution. AMs are designed and evolved to complement existing metabolic pathways. Specifically, to form the valine precursor α-ketovaline in an auxotrophic Escherichia coli knockout strain. This project is a proof of principle and blueprint for the development of AM-based tools for studying cellular regulation to answer basic biological questions.
Interactions, dynamics and functionality at nanoscale characterized by confocal laser scanning microscopy and fluorescence correlation spectroscopy Research Project | 5 Project MembersFluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and Förster resonance energy transfer (FRET) play an irreplaceable role to determine the number and movement of molecules, biological entities, and nanoparticles in biological or synthetic systems. These methods can analyze particles, not only in solution, but also attached to surfaces, inside cells, or nanoparticles. From these measurements, the number of particles, binding strength (KD), distance, surface modifications, and the speed of particles can be determined. When coupled with confocal laser scanning microscopy (CLSM), the particles can be visualized in various environments as well. We require financial support for a new state of the art CLSM/FCS/FCCS instrument in the Department of Chemistry at the University of Basel that will include all three spectroscopy modes (FCS, FCCS and FRET) as well as a high resolution confocal microscope and cell incubation platform. We strongly depend on these techniques in our on-going and future projects. This instrument will allow for resolving images of fluorescently labeled particles from the nano to micrometer size (~140 nm- 2 µm), as well as enable the long term study of particles inside growing cells. FCS and FCCS will allow for measurements of product formation and molecule localization at the site of interest, as well as allow for the determination of binding, release, and movement of particles. It will also expand our capabilities by giving us the ability to detect samples inside growing cells for extended periods of time, which was previously not possible. The new software will make data analysis and interpretation much simpler, and with models adapted for complex systems. Finally, the system is designed in a modular fashion allowing for the future upgrade with more diverse laser arrays as well as the addition of enhanced fluorescence lifetime imaging microscopy. This new state of the art CLSM/FCS/FCCS instrument will support research emphasized by the University of Basel, within the scope of "life- and nanoscience. It will ensure the international competitiveness of the research groups involved in the proposal (groups from the Department of Chemistry, Biozentrum, and the Swiss Tropical and Public Health Institute), and their collaborations within the NCCR Molecular Systems Engineering, the Swiss Nanoscience Institute, and industrial partners. Moreover, other groups from adjacent departments of the University of Basel and various industrial partners can have access based on joint projects.
Picosecond Transient Absorption Setup for Detection of Short-Lived Photoproducts and Excited States in Molecular Systems Research Project | 6 Project MembersMany photophysical and photochemical processes which are relevant for light-to-chemical energy conversion occur on very rapid timescales. Time-resolved UV-Vis absorption spectroscopy has become an indispensable tool in modern photochemistry. Several ongoing Ph. D. theses and postdoctoral research projects in the main applicant's group ask for a transient absorption spectrometer with picosecond time resolution and an appropriate laser source. Among these projects are for example the investigation of photoinduced multi-electron transfer reactions in order to spatially separate multiple electrons from multiple holes, which is of key importance for producing chemical fuels with sunlight as energy input (projects 1 and 2). Similarly, picosecond transient absorption spectrosocopy will permit mechanistic studies of photoinduced proton-coupled electron transfer (PCET) reactions which will greatly further our current fundamental understanding of this important class of reactions (project 3). The activation of small inert molecules such as H 2 O, CO 2 or N 2 will invariably rely on multi-electron, multi-proton chemistry hence the proposed photochemical studies are important in the greater context of solar energy conversion.
NCCR Molecular Systems Engineering Research Project | 33 Project MembersThe NCCR Molecular Systems Engineering combines competences from life sciences, chemistry, physics, biology, bioinformatics and engineering sciences. More than 100 researchers and support personnel distributed into four work packages and 31 projects work together to address systems engineering challenges by integrating novel chemical and biological modules into molecular factories and cellular systems for the production of high added-value chemicals and applications in medical diagnostics and treatment. Molecular systems engineering relies on the combination of both chemical- and biological modules. In this approach, complex dynamic phenomena emerge as the result of the integration of molecular modules designed to interact in a programmed way with their complex environment. In this manner, it should be possible to create molecular factories and cellular systems whose properties are more than the sum of the attributes of the individual modules. The commitments of the leading houses, the University of Basel and ETH Zurich, also include new (joint) professorships, and extensive training of a new generation of scientists and technologists, leading to a long-term paradigm shift in molecular sciences and a new structure of the Swiss research landscape.
NCCR MSE - Molecular Factories Based on Artificial Metalloenzymes Research Project | 3 Project MembersArtificial metalloenzymes (ArMs) result from the incorporation of abiotic cofactors within a host protein. In collaboration with various groups of the NCCR, we aim at engineering metabolic pathways incorporating ArMs. The challenges addressed during this funding period include: i) successful compartmentalization and evolution of ArMs within the periplasm of E coli, ii) engineering enzyme cascades with ArMs and iii) cross-regulation of ArMs.
Artificial Metalloenzymes: Supramolecular Anchoring and Activation Research Project | 1 Project MembersLife emerges from inanimate matter thanks to a complex network of tightly cross-regulated enzymatic reaction cascades. To mimick this, we have emplemented the cross-regulation of an artificial imine reductase which is turned on in the presence of a protease, generating a tripeptide that acts as a cross-regulator for the imine reductase.
Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications Research Project | 1 Project MembersArtificial metalloenzymes result from combining a catalytically competent organometallic moiety with a host protein. The resulting hybrid catalyst combine attractive features of both chemo- and biocatalysts. In recent years, the Ward group has exploited the biotin-streptavidin towards the creation of artificial metalloenzymes for hydrogenation, allylic alkylation, sulfoxidation, alcohol oxidation, dihydroxylation, transfer-hydrogenation and olefin metathesis. The latter two systems were shown to be particularly stable towards E. coli cellular extracts. Within this funding period, it is proposed to exploit this finding towards the implementation of directed evolution protocols for the optimization of the performance of artificial metalloenzymes. Four complementary and intedisciplinary sub-projects will be investigated: i) exploiting streptavidin expressed in the periplasm; ii) cascade reactions with artificial metalloenzymes; iii) optimization of artificial transfer-hydrogenase for the production of high-added value amines and aminoacids and iv) directed evolution of artificial metathesases. i) In order to circumvent the inhibition of the biotinylated precious metal catalyst by glutathione (present in milimolar amounts in the cytoplasm), it is proposed to target streptavidin to the periplasm. This will allow us to sidestep the lengthy purification of streptavidin prior to catalysis, eventually allowing the implementation of directed evolution protocols. ii) We have shown that artificial metalloenzymes are compatible with a variety of biocatalysts. Combining artificial metalloenyzmes with natural enzymes will lead to complex reaction cascades that can be used a) as a high-throughput colorimetric assay or b) to complement metabolic pathways. iii) The above developments will be exploited towards the preparation of a) high-added value amines via the enantioselective imine reduction and b) leucine by relying on a selection strategy based on E. coli leucine auxotrophs. iv) Thanks to the inertness of artificial metathesases based on the biotin-streptavidin technology, the performance of these will be optmized using crude E. coli cell extracts. For this purpose, we will rely screening a fluorophore-quencher substrate which, upon ring closing metathesis releases the quencher, thus becoming fluorescent. Ultimately, we aim at developing artificial metalloenzymes that outperform classical organometallic catalysts. In a biomimetic spirit and thanks to Darwinian protocols, we anticipate that the presence of an optimized second coordination sphere provided by the protein environment will allow to achieve this ambitious goal.
