Brustad, E.M.; Nicewicz, D.A.

ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362

A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

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.