Evolving Artificial Metalloenzymes via Random Mutagenesis
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.
Reaction: CyclopropanationMax TON: 66ee: 94Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: N-H InsertionMax TON: 73ee: 40Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: S-H insertionMax TON: 64ee: 32Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: Si-H insertionMax TON: 35ee: 64Notes: Mutagenesis of the ArM by error-prone PCR
Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme
Angew. Chem. Int. Ed. 2021, 60, 15063-15068, 10.1002/anie.202103437
Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ-methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N–H insertion, whereas variants with reduced Eo values gave higher S–H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.
Reaction: CyclopropanationMax TON: ---ee: >99PDB: ---Notes: yield: styrene cyclopropanation 71% max, cf free heme <5%
Reaction: N-H InsertionMax TON: ---ee: ---PDB: ---Notes: Yield: aniline insertion 74-93%
Reaction: S-H insertionMax TON: ---ee: ---PDB: ---Notes: Yield: thiophenol insertion 18-36% but still outperforms heme