Asymmetric reductive amination of ketones using ω-transaminases (ω-TAs) offers a promising alternative to the chemocatalytic synthesis of chiral amines. One fundamental challenge to the biocatalytic strategy is the very low enzyme activities for most ketones compared with native substrates (i.e., <1% relative to pyruvate). Here we have demonstrated that a single point mutation in the active site of the (S)-selective ω-TA from Ochrobactrum anthropi could induce a remarkable acceleration of the amination reaction without any loss in stereoselectivity and enzyme stability. Molecular modeling of quinonoid intermediates, alanine scanning mutagenesis and kinetic analysis revealed that the W58 residue acted as a steric barrier to binding and catalytic turnover of ketone substrates. Removal of the steric strain by W58L substitution, which was selected by partial saturation mutagenesis, led to dramatic activity improvements for structurally diverse ketones (e.g., 340-fold increase in kcat/KM for acetophenone). The W58L mutant afforded an efficient synthesis of enantiopure amines (i.e., >99% ee) using isopropylamine as an amino donor.
Bibliographical notePublisher Copyright:
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
All Science Journal Classification (ASJC) codes
- Organic Chemistry