A General Method for Artificial Metalloenzyme Formationthrough Strain-Promoted Azide–Alkyne Cycloaddition
ChemBioChem 2014, 15, 223-227, 10.1002/cbic.201300661
Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both SiH and olefin insertion reactions involving these carbene precursors.
Ligand type: Poly-carboxylic acidHost protein: tHisFOptimization: ---Reaction: CyclopropanationMax TON: 81ee: ---PDB: 1THFNotes: ---
Ligand type: Poly-carboxylic acidHost protein: tHisFOptimization: ---Reaction: Si-H insertionMax TON: 7ee: ---PDB: 1THFNotes: ---
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