Rate-Determining Step and Enantioselectivity Divergence in Nonheme Fe Enzyme-Catalyzed Nitrogen Migration: Directed Evolution of Isopenicillin N Synthases for Asymmetric Non-Canonical Amino Acid Synthesis

We describe the study and engineering of nonheme Fe enzymes as highly efficient biocatalysts for 1,3-nitrogen migration reactions, enabling the enantioselective synthesis of non-canonical amino acids, which represent valuable building blocks in pharmaceuticals, peptide therapeutics, and asymmetric synthesis. Nonheme Fe enzyme Isopenicillin N synthase from Emericella nidulans (EniIPNS) was repurposed and evolved into a quadruple mutant (V185L I187V S102I R279H, IPNSNim), enabling the conversion of a range of azanyl esters into N-protected L-amino acids with enantiopreference opposite to our previously engineered D-amino acid producing 1-aminocyclopropane-1-carboxylate oxidase (ACCONim). Notably, IPNSNim achieved a total turnover number of 16,700, surpassing state-of-the-art small-molecule Fe catalysts by 340-fold and representing the highest TTN reported for a nonheme Fe enzyme in a new-to-nature reaction. Together with ACCONim, this platform enabled access to either enantiomer of non-canonical amino acids with high efficiency. Mechanistic studies using deuterium-labeled substrates indicated that, in contrast to ACCONim, the hydrogen atom transfer (HAT) step is not the rate-limiting step in IPNS-catalyzed nitrogen migration reaction. Moreover, unlike ACCO, IPNS confers excellent enantiocontrol over the HAT event. Combined computational and experimental studies shed light on the origin of enantiodivergence, unraveling a rare mechanistic and enantioselectivity dichotomy in asymmetric nitrogen migration catalyzed by two phylogenetically related enzymes IPNS and ACCO.