Construction of dual-cofactor artificial metalloenzymes for synergistic and enantiodivergent catalysis of Michael addition reactions

Artificial metalloenzymes (ArMs) offer a promising tool for catalytic organic synthesis with abiotic activities. Although synergistic catalysis has been widely applied in small-molecule systems, it has rarely been implemented in ArMs, in part owing to the challenge of assembling synergistic cofactors within a protein scaffold. Here we report the modular synthesis of dual-cofactor ArMs incorporating a biotinylated nickel-based cofactor and a peptide cofactor, both of which are anchored within neighbouring subunits of the homotetrameric streptavidin. The resulting synergistic ArM catalysed the asymmetric Michael addition of ketones to enals, and its catalytic performance was improved by chemo-genetic optimization. The optimization utilized high-throughput solid-phase peptide synthesis. Mechanistic studies revealed the molecular basis of the synergistic mechanism, as well as the role of key mutations in stabilizing the cofactors and active site geometry. Notably, we developed two complementary ArM systems that enable enantiodivergent synthesis of chiral building blocks. This work establishes a general strategy for the synthesis of ArMs with synergistic cofactors and expands the scope of biocatalytic asymmetric synthesis.