Semi-rational design of an (S)-selective ω-transaminase for the asymmetric synthesis of the bulky aryl-alkyl chiral amine (S)-3-methyl-1-phenylbutylamine.

ω-Transaminases (ω-TAs) provide an efficient route to the asymmetric synthesis of chiral amines. However, the intrinsic steric constraints of native ω-TA active sites lead to extremely low or undetectable catalytic activity toward bulky aryl-alkyl substrates. Here, we combined a substrate-truncation strategy with semi-rational design to engineer an (S)-selective ω-TA for the asymmetric synthesis of the bulky aryl-alkyl chiral amine (S)-3-methyl-1-phenylbutylamine. Using acetophenone as a truncated substrate, we identified an (S)-selective ω-TA, LMG45, from Pseudomonas putida. Starting from this enzyme, alanine scanning of the active pocket yielded the mutant M1 (L60A), which showed the first detectable activity toward the target substrate isovalerophenone. Subsequently, active pocket remodeling, dynamic cross-correlation matrix (DCCM)-guided cooperative evolution, and substrate tunnel engineering yielded the mutant M5 (L60A/W61L/N87P/Q91G/I413A) with improved catalytic activity. Further saturation mutagenesis at R415 afforded the mutant M6 (M5-R415V) with 72% conversion and 99.5% ee in the asymmetric synthesis of (S)-3-methyl-1-phenylbutylamine. The mutant M6 not only exhibited enhanced catalytic activity toward substrates already accepted by the wild-type enzyme (WT) but also showed activity toward five bulky substrates that were not accepted by WT. This work provides a promising biocatalyst for the synthesis of bulky aryl-alkyl chiral amines.