Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis

Artificial metalloenzymes result from anchoring an organometallic catalyst within evolvable protein scaffold. Thanks to its dimer of dimers quaternary structure, streptavidin allows the precise positioning two metal cofactors activate a single substrate, thus expanding reaction scope accessible artificial metalloenzymes. To validate this concept, we report herein on our efforts engineer and evolve hydroaminase based dual gold activation alkynes. Guided by modelling, designed chimeric equipped with hydrophobic lid shielding active site, which enforces advantageous synergistic biotinylated cofactors. Three rounds directed evolution using Escherichia coli cell-free extracts led identification mutants favouring either anti-Markovnikov product (an indole carboxamide 96% regioselectivity, 51 turnover numbers), resulting ?,?-activation ethynylphenylurea or Markovnikov (a phenyl-dihydroquinazolinone 99% 333 ?-activation alkyne gold. Dual catalysis is widely employed natural functionalize challenging substrates. Now, concept applied designing enabling unnatural mechanism terminal