Characterization and computation-supported engineering of an ω-transaminase

An (S)-selective homodimeric ω-transaminase from Pseudomonas jessenii (PjTA) that naturally converts 6-aminohexanoic acid to 6-oxohexanoic acid in the caprolactam biodegradation pathway was discovered and characterized in our lab. However, the applications of dimeric ω-transaminases were hindered by enzyme instability and their limited substrate scope. Thus PjTA was selected as the model system and protein engineering was used to improve the enzyme stability and redirect the preference of substrate. A computational method (FRESCO) which was previously explored by our group was applied to stabilize PjTA. Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for the stability of PjTA. After a rational combination, a robust PjTA was obtained that more active at high temperatures and more tolerant to cosolvents and to a high concentration of amino donor (isopropylamine). Then this robust PjTA was selected to redesign the enzyme binding pockets to improve the production of enantiopure bulky amines from prochiral ketones via asymmetric synthesis. A computational approach with a high success rate was explored and the yields in the synthesis of selected bulky amines were highly improved with >99% enantiomeric excess