Project

Discipline

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

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

Active participation

Title	Date	Number	Inventor	Owner

Number	Title	Start	Funding scheme

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.