Abstract
We describe the reprogramming and directed evolution of nonheme Fe enzyme isopenicillin N synthase (IPNS) as an efficient biocatalyst for 1,3-nitrogen migration reactions via an unnatural mechanism. Directed evolution of isopenicillin N synthase from Emericella nidulans furnished a quadruple mutant (EniIPNS V185L I187V S102I R279H, IPNS(Nim)), enabling the conversion of a range of azanyl esters into N-protected l-arylglycines. IPNS(Nim) achieved a TTN of 16 000 and a TOF of 1200 min(-1). This TTN surpassed state-of-the-art small-molecule Fe catalysts by 330-fold and represented the highest TTN value reported for a nonheme Fe enzyme in a new-to-nature reaction. IPNS(Nim) and our previously evolved ACCO(Nim) (ACCO: 1-aminocyclopropane-1-carboxylic acid oxidase) exhibited complementary enantiopreference, allowing enantioselective synthesis of either l- or d-arylglycines-essential building blocks in clinically important peptide therapeutics. Mechanistic studies revealed a biocatalyst-controlled switch in the rate-determining step (RDS): While the hydrogen atom transfer (HAT) step is the RDS for ACCO(Nim)-catalyzed nitrogen migration, it is likely not with IPNS(Nim). Moreover, while ACCO(Nim) exhibits almost no enantioselectivity in this HAT step, IPNS(Nim) confers excellent enantiocontrol over HAT. Computational studies using density functional theory calculations and molecular dynamics simulations suggested that IPNS and ACCO adopt two different substrate binding modes. Classical MD simulations shed light on important interactions between the substrate and active-site residues that control the substrate binding mode and enantioselectivity.