High-throughput screening of artificial metalloenzymes based on the streptavidin technology.

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Markus Jeschek, Thomas R. Ward, Sven Panke

ETH Zurich, Switzerland

Due to the extraordinary strength of its non-covalent bond to biotin (KD < 10-13 M) streptavidin can be used as a handy and flexible tool in synthetic biology including the use as a protein scaffold for the creation of artificial metalloenzymes that are able to perform bio-orthogonal reactions including transfer hydrogenation, allylic alkylation or enantioselective sulfoxidation. In this approach an organometallic catalyst is coupled to a biotin moiety to enable the supramolecular anchoring of the catalytic unit into the protein scaffold of the biotin binding pocket of streptavidin in order to combine the features of homogenous catalysis with the beneficial characteristics of enzymes[1]. The resulting complex can exhibit superior catalytic features compared to the free low-molecular catalyst itself. These features potentially include enhanced stereoselectivity, access to aqueous media and ambient conditions, catalytic rate acceleration, higher turnover numbers, catalyst recovery and protection from degradation and so forth. Additionally to chemical tailoring of the biotinylated catalysts an optimization of the protein scaffold can be achieved by the means of protein engineering. This has been shown for several reactions and led to a significant improvement of the catalytic performance[2]. Currently we are working on the development of a fluorescence based assay for artificial metalloenzymes that will allow for the efficient screening of beneficial mutants in a high-throughput manner. We hope that this will lead to the development of a broad range of artificial metalloenzymes and generate a bio-orthogonal tool box that could be employed in chemical biology as well as synthetic biology. [1] Ward TR. Acc Chem Res. 2011. 44(1):47-57. [2] Dürrenberger et al. Angew Chem Int Ed Engl. 2011. 50(13):3026-9.