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

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.

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