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.

Barker, P.D.; Boss, S.R.

Angew. Chem. Int. Ed. 2021, 60, 10919-10927, 10.1002/anie.202015834

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6-arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

Golinelli-Pimpaneau, B.

PLoS One 2012, 7, e51128, 10.1371/journal.pone.0051128

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

Berggren, G.; Lindblad, P.

Energy Environ. Sci. 2018, 11, 3163-3167, 10.1039/C8EE01975D

[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.

Barker, P.D.; Boss, S.R.

Johnson Matthey Technol. Rev. 2020, 64, 407-418, 10.1595/205651320x15928204097766

Generation of artificial metalloenzymes (ArMs) has gained much inspiration from the general understanding of natural metalloenzymes. Over the last decade, a multitude of methods generating transition metal-protein hybrids have been developed and many of these new-to-nature constructs catalyse reactions previously reserved for the realm of synthetic chemistry. This perspective will focus on ArMs incorporating 4d and 5d transition metals. It aims to summarise the significant advances made to date and asks whether there are chemical strategies, used in nature to optimise metal catalysts, that have yet to be fully recognised in the synthetic enzyme world, particularly whether artificial enzymes produced to date fully take advantage of the structural and energetic context provided by the protein. Further, the argument is put forward that, based on precedence, in the majority of naturally evolved metalloenzymes the direct coordination bonding between the metal and the protein scaffold is integral to catalysis. Therefore, the protein can attenuate metal activity by positioning ligand atoms in the form of amino acids, as well as making non-covalent contributions to catalysis, through intermolecular interactions that pre-organise substrates and stabilise transition states. This highlights the often neglected but crucial element of natural systems that is the energetic contribution towards activating metal centres through protein fold energy. Finally, general principles needed for a different approach to the formation of ArMs are set out, utilising direct coordination inspired by the activation of an organometallic cofactor upon protein binding. This methodology, observed in nature, delivers true interdependence between metal and protein. When combined with the ability to efficiently evolve enzymes, new problems in catalysis could be addressed in a faster and more specific manner than with simpler small molecule catalysts.

Clark, D.S.; Hartwig, J.F.; Keasling, J.D.; Mukhopadhyay, A.

Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.