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.

Lewis, J.C.

Nat. Chem. 2018, 10, 318-324, 10.1038/nchem.2927

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N–H, S–H and Si–H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.