Borovik, A.S.; Hendrich, M.P.; Moënne-Loccoz, P.

J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Albrecht, M.; Paradisi, F.

Angew. Chem. Int. Ed. 2018, 130, 10837-10842, 10.1002/ange.201807168

Im Tausch gegen NHC: Die Einfügung eines N‐heterocyclischen Carbenliganden (grün/blau) als Ersatz für His in das aktive Zentrum des Redoxenzyms Azurin rekonstituiert das T1‐Kupferzentrum. Der resultierende Komplex ist spektroskopisch kaum unterscheidbar von der N‐Bindung von His oder N‐Methylimidazol, senkt aber signifikant das Reduktionspotential des Kupferzentrums und erleichtert dadurch Elektronentransferprozesse.

Jäger, C.M.; Pordea, A.

Faraday Discuss. 2022, 10.1039/d1fd00070e

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.

Jaussi, R.

Protein Expression Purif. 2014, 93, 54-62, 10.1016/j.pep.2013.10.015

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650 mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2 molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.