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Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications

English title Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications
Applicant Ward Thomas R.
Number 144354
Funding scheme Project funding
Research institution Institut für Anorganische Chemie Universität Basel
Institution of higher education University of Basel - BS
Main discipline Inorganic Chemistry
Start/End 01.10.2012 - 30.09.2015
Approved amount 620'000.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Organic Chemistry

Keywords (7)

biotin-avidin technology; enzyme cascade; artificial metalloenzymes; transfer-hydrogenation; directed evolution; metathesis; organometallic chemistry

Lay Summary (English)

Lead
Lay summary

Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications

Lay Summary

"Catalysis is the concept which best captures the Spirit of Chemistry: the Miracle of consumption and regeneration."

In a catalytic cycle, a molecule, the catalyst, intimately participates to the reaction by accelerating a chemical transformation but is not consumed. Such catalysts have enormous societal impact, significantly decreasing the energy (and thus cost) required to produce various products, ranging from the delicate process of digestion, to fertilizers sustaining life on earth.

Natural enzymes have evolved over millions of years to orchestrate the ballet of life at a minimal energetic cost. A human being only requires 2000 Watts to sustain his daily activities. For this, we rely on 30'000 different proteins, most of which acting as enzymes.

To address the requirements for the production of various chemicals, ranging from paints, pharmaceuticals, fertilisers, foods etc., different types of catalysts are used: enzymes, homogeneous- and heterogeneous catalysts. All three types offer advantages and disadvantages.

With the aim of exploiting their most attractive and complementary features, it is proposed to merge homogeneous and enzymatic catalysis to yield artificial metalloenzymes. As we have demonstrated in the past, incorporation of an active metal moiety within a protein environment affords hybrid catalysts with very promising properties, including high activities and selectivities, reminiscent of both homogeneous and enzymatic catalysts.

Random mutations on an enzyme’s gene leads to a slight modification its the catalytic properties. Thanks to rounds of mutation and selection for improved catalytic performance, current enzymes approach perfection. In stark contrast, homogeneous- and heterogeneous catatlyst often display poorer catalytic properties as they cannot be evolved using Darwinian schemes (i.e. directed evolution).

Within this proposal, we aim at implementing such directed evolution schemes towards the optimization of artificial metalloenzymes. With this goal in mind, two reactions were selected: transfer hydrogenation and olefin metathesis. These reactions have been selected as we have shown that they i) are amenable to directed evolution schemes and ii) complement natural enzymes in terms of reactivity. Ultimately, we propose that these reactions may be used within a cellular environment to complement the metabolism.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Artificial metalloenzymes for the diastereoselective reduction of NAD(+) to NAD(2)H
Quinto T., Haussinger D., Kohler V., Ward T. R. (2015), Artificial metalloenzymes for the diastereoselective reduction of NAD(+) to NAD(2)H, in Org. Biomol. Chem., 357-60.
Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding
Fujieda N., Schatti J., Stuttfeld E., Ohkubo K., Maier T., Fukuzumi S., Ward T. R. (2015), Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding, in Chem. Sci., 6, 4060-4065.
Evaluation of the Formate Dehydrogenase Activity of Three-Legged Pianostool Complexes in Dilute Aqueous Solution
Keller S. G., Ringenberg M. R., Haussinger D., Ward T. R. (2015), Evaluation of the Formate Dehydrogenase Activity of Three-Legged Pianostool Complexes in Dilute Aqueous Solution, in European Journal of Inorganic Chemistry, 5860-5864.
Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design
Heinisch T., Pellizzoni M., Durrenberger M., Tinberg C. E., Kohler V., Klehr J., Haussinger D., Baker D., Ward T. R. (2015), Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design, in J. Am. Chem. Soc., 137, 10414-9.
Latest Developments in Metalloenzyme Design and Repurposing
Heinisch T., Ward T. R. (2015), Latest Developments in Metalloenzyme Design and Repurposing, in European Journal of Inorganic Chemistry, 3406-3418.
Profluorescent substrates for the screening of olefin metathesis catalysts
Reuter R., Ward T. R. (2015), Profluorescent substrates for the screening of olefin metathesis catalysts, in Beilstein J. Org. Chem., 1886-1892.
High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis
Nogueira E. S., Schleier T., Durrenberger M., Ballmer-Hofer K., Ward T. R., Jaussi R. (2014), High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis, in Prot. Expr. Purif., 54-62.
Neutralizing the Detrimental Effect of Glutathione on Precious Metal Catalysts
Wilson Y. M., Durrenberger M., Nogueira E. S., Ward T. R. (2014), Neutralizing the Detrimental Effect of Glutathione on Precious Metal Catalysts, in J. Am. Chem. Soc., 136, 8928-8932.
Recent achievments in the design and engineering of artificial metalloenzymes
Durrenberger M., Ward T. R. (2014), Recent achievments in the design and engineering of artificial metalloenzymes, in Current Opinion in Chemical Biology, 19, 99-106.
Recent Trends in Biomimetic NADH Regeneration
Quinto T., Kohler V., Ward T. R. (2014), Recent Trends in Biomimetic NADH Regeneration, in Topics in Catalysis, 57, 321-331.
Structural, Kinetic, and Docking Studies of Artificial imine Reductases Based on Biotin-Streptavidin Technology: An Induced Lock-and-Key Hypothesis
Robles V. M., Durrenberger M., Heinisch T., Lledos A., Schirmer T., Ward T. R., Marechal J. D. (2014), Structural, Kinetic, and Docking Studies of Artificial imine Reductases Based on Biotin-Streptavidin Technology: An Induced Lock-and-Key Hypothesis, in J. Am. Chem. Soc., 15676-15683.
A Dual Anchoring Strategy for the Localization and Activation of Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology
Zimbron J. M., Heinisch T., Schmid M., Hamels D., Nogueira E. S., Schirmer T., Ward T. R. (2013), A Dual Anchoring Strategy for the Localization and Activation of Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology, in J. Am. Chem. Soc., 135, 5384-5388.
Fluorescence-Based Assay for the Optimization of the Activity of Artificial Transfer Hydrogenase within a Biocompatible Compartment
Heinisch T., Langowska K., Tanner P., Reymond J. L., Meier W., Palivan C., Ward T. R. (2013), Fluorescence-Based Assay for the Optimization of the Activity of Artificial Transfer Hydrogenase within a Biocompatible Compartment, in ChemCatChem, 720-723.
Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin-Streptavidin Technology
Schwizer F., Kohler V., Durrenberger M., Knorr L., Ward T. R. (2013), Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin-Streptavidin Technology, in ACS Catal., 1752-1755.
Metal-Catalyzed Organic Transformations inside a Protein Scaffold Using Artificial Metalloenzymes
Praneeth V. K. K., Ward T. R. (2013), Metal-Catalyzed Organic Transformations inside a Protein Scaffold Using Artificial Metalloenzymes, in Coordination Chemistry in Protein Cages: Principles, Design, and Applications, 203-219.
Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes
Kohler V., Wilson Y. M., Durrenberger M., Ghislieri D., Churakova E., Quinto T., Knorr L., Haussinger D., Hollmann F., Turner N. J., Ward T. R. (2013), Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes, in Nat. Chem., 93-99.
Biotinylated Rh(III) Complexes in Engineered Streptavidin for Accelerated Asymmetric C-H Activation
Hyster T. K., Knorr L., Ward T. R., Rovis T. (2012), Biotinylated Rh(III) Complexes in Engineered Streptavidin for Accelerated Asymmetric C-H Activation, in Science, 500-503.

Collaboration

Group / person Country
Types of collaboration
Prof. David Baker, Univ. Washington Seattle United States of America (North America)
- Publication
Prof. Tom Rovis, CSU Fort-Collins United States of America (North America)
- Publication
Prof. Sven Panke, DBSSE, ETHZ at Basel Switzerland (Europe)
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
PSI Individual talk Artificial Metalloenzymes: Challenges and Opportunities 27.10.2015 Villigen, Switzerland Ward Thomas R.;
MS Topical Seminar on Catalytic Chemistry in Living Cells Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 17.09.2015 Groningen, Netherlands Ward Thomas R.;
162. Synthetic Biology Talk given at a conference Artificial Metalloenzymes for Synthetic Biology Applications 03.09.2015 London, Great Britain and Northern Ireland Ward Thomas R.;
ACS Talk given at a conference Optimization of the performance of artficial metalloenzymes by fine-tuning of the second coordination sphere 16.08.2015 Boston, MA, United States of America Ward Thomas R.;
GRC Synthetic Biology Talk given at a conference Artificial Metalloenzymes for Synthetic Biology Applications 29.06.2015 Newry, ME, United States of America Ward Thomas R.;
ISMEC 2015 Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 25.06.2015 Wroclaw, Poland Ward Thomas R.;
Metacode Meeting Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 17.06.2015 Dresden, Germany Ward Thomas R.;
ACIB Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 11.06.2015 Graz, Austria Ward Thomas R.;
Transam 2 Talk given at a conference Artificial Metalloenzymes for the Synthesis of Chiral Amines and Derivatives 05.03.2015 Greifswald, Germany Ward Thomas R.;
Gordon Research Symposium, Metals in Biology Talk given at a conference Manipulating Metals with Biology 30.01.2015 Ventura, CA, United States of America Ward Thomas R.;
UCI Individual talk Artificial Metalloenzymes: Challenges and Opportunities 23.01.2015 Irvine, United States of America Ward Thomas R.;
Biotrans Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 23.01.2015 Wien, Austria Ward Thomas R.;
ISIS Summer School on Supramolecular Chemistry Individual talk Artificial Metalloenzymes: Challenges and Opportunities 09.09.2014 Strasburg, France Ward Thomas R.;
Eurobic 12 Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 25.08.2014 Zürich, Switzerland Ward Thomas R.;
KAUST Individual talk Artificial Metalloenzymes: Challenges and Opportunities 02.06.2014 Thuwal, Saudi Arabia Ward Thomas R.;
Trends in Bioinorganic Chemistry Individual talk Artificial Metalloenzymes: Challenges and Opportunities 22.05.2014 Lund, Sweden Ward Thomas R.;
CHARM3AT Talk given at a conference Artificial Metalloenzymes: Recent Progress and Challenges 28.04.2014 Gif-sur-Yvette, France Ward Thomas R.;
Caltech Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 03.01.2014 Pasadena, CA, United States of America Ward Thomas R.;
ENSI Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 13.12.2013 Caen, France Ward Thomas R.;
Uppsala University Individual talk Artificial Metalloenzymes: Challenges and Opportunities 26.11.2013 Uppsala, Sweden Ward Thomas R.;
COST CM 1005 Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 11.11.2013 Valletta, Malta Ward Thomas R.;
International Bielefeld CeBiTech Research Conference Talk given at a conference Engineering Reaction Cascades with Artificial Metalloenzymes 23.09.2013 Bielefeld, Germany Ward Thomas R.;
XXVI RSEQ Biennial Meeting Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 16.09.2013 Santander, Spain Ward Thomas R.;
ICBIC Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 26.07.2013 Grenoble, France Ward Thomas R.;
ISOM XX Talk given at a conference In Vivo Screening of Artificial Metalloenzymes for Olefin Metathesis : Challenges and Opportunities 16.07.2013 Nara, Japan Ward Thomas R.;
Université de Strasbourg Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 13.06.2013 Strasburg, France Ward Thomas R.;
Foldamers Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 10.04.2013 Paris, France Ward Thomas R.;
NCCC XIV Talk given at a conference Artificial Metalloenzymes: Challenges and Opportunities 12.03.2013 Leeuwenhorst, Netherlands Ward Thomas R.;
University of Washington Individual talk Artificial Metalloenzymes: Challenges and Opportunities 28.01.2013 Seattle, United States of America Ward Thomas R.;
Scripps Research Institute Individual talk Artificial Metalloenzymes: Challenges and Opportunities 17.01.2013 La Jolla, CA, United States of America Ward Thomas R.;
University of Marburg, Fachbereich Chemie Individual talk Artificial Metalloenzymes Based on the Biotin Avidin Technology: Challenges and Recent Advances 14.01.2013 Marburg, Germany Ward Thomas R.;
3rd Metacode Meeting Talk given at a conference Artificial Metalloenzymes Progress, Challenges and Recent Advances 12.11.2012 Basel, Switzerland Ward Thomas R.;
EMBO conference Talk given at a conference Artificial Metalloenzymes Based on the Biotin Avidin Technology 09.10.2012 Groningen, Netherlands Ward Thomas R.;


Patents

Title Date Number Inventor Owner
Method for neutralizing detrimental effects of thiol-bearing compounds on metal catalysts 05.11.2014 EP 14170960.0 - 1352
Novel process to produce streptavidin and other biotin-binding proteins 05.11.2013 EP13178560.2

Associated projects

Number Title Start Funding scheme
162348 Broadening the Scope of Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology 01.10.2015 Project funding
163996 Interactions, Dynamics, and Functionality at Nanoscale Characterised by Confocal Laser Scanning Microscopy and Fluorescence Correlation Spectroscopy 01.12.2015 R'EQUIP
126366 Designed Evolution of Artificial Metalloenzymes Based on Streptavidin or Carbonic Anhydrase as Protein Scaffold 01.10.2009 Project funding
157687 Picosecond Transient Absorption Setup for Detection of Short-Lived Photoproducts and Excited States in Molecular Systems 01.11.2015 R'EQUIP

Abstract

Artificial 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.
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