Work

Enhancing ω-transaminase catalysis of bulky substrates guided by binding mechanism

Year: 2026

Type: article

Abstract: Chiral amines are indispensable building blocks in pharmaceutical synthesis. (R)-3-([1,1′-biphenyl]-4-yl)-2-aminopropan-1-ol (ABP), a key chiral amine intermediate of the drug Entresto, has remained difficult to access via enzymatic asymmetric synthesis due to the inability of ω-transaminases to accommodate its bulky ketone precursor. Here, we engineer an ω-transaminase from Bacillus megaterium by integrating Funnel metadynamics, PathDetect-SOM, and path collective variables to reconstruct the free energy surface of substrate binding, pinpointing high-energy barrier regions of the binding process and the trade-off between tunnel accessibility and productive binding. Dynamic cross-correlation network analysis further identifies distal regulatory sites governing these bottleneck regions. Iterative experimental optimization yields the 13M variant, exhibiting catalytic activity 106 times that of the wild type with >99% enantiomeric excess, enabling the efficient enzymatic asymmetric synthesis of ABP. This integrated computational-experimental framework for resolving the tunnel-pocket binding trade-off offers a generalizable strategy for engineering ω-transaminases toward bulky substrates in green pharmaceutical synthesis. This study combines enhanced sampling with experiments to reveal how bulky substrates enter and bind to ω-transaminases. Distal-site engineering balanced tunnel access and binding stability, yielding 106-fold activity and >99% selectivity.

Source: Nature Communications

DOI: 10.1038/s41467-026-78636-0