Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Abstract

Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

“…Directed evolution has proven to be a powerful strategy for protein engineering, especially with regard to improvements of protein activity and thermostability (Ruan et al, 2022;Su et al, 2022). This process involves the generation of a vast mutant library of the gene of interest through random mutagenesis techniques (e.g., error-prone Polymerase Chain Reaction [epPCR] or DNA shuffling), which are then screened to identify mutants that enhance enzymatic performance in nonnatural environments (Athavale et al, 2022). The efficient generation of random mutations in the target gene is an important step in directed evolution.…”