Strengthening core-region hydrogen-bond networks and rigidifying surface loop to enhance thermostability of an (R)-selective transaminase converting chiral hydroxyl amines

Transaminases have important applications in the synthesis of drug intermediates such as chiral amines. However, natural transaminases exhibit suboptimal thermal stability, limiting their further applications. Building upon an Rhodobacter sp.-derived (R)-selective transaminase (RbTA), we report a dual-region coupling engineering approach to improve thermostability of RbTA by strengthening the core hydrogen-bond networks and rigidifying the flexible surface loop. Through single strategy, we identified 4 thermostability improved single mutations, among which I249Q demonstrated the most substantial improvement, achieving a 18-fold increase in half-life (t1/240) and a 11.2 ℃ increase in T5010. Then in strategic coupling, the synergistic effect of dual-region modification was observed in both thermal stability and activity enhancement, as mutant with the best high-temperature catalytic performance, R136P/F228Y, had its T5010 improved by 7.1℃ and exhibited a 4.2-fold increase in kcat/Km towards (R)-3-amino-1-butanol. Finally, R136P/F228Y achieved a 20.5 % improvement in conversion over WT in an analytical-scale synthesis in 72 h at a 5 ℃ elevated catalytic temperature. Molecular dynamics simulations demonstrated that the synergy of the formation of new hydrogen bonds and decrease in flexibility accounted for the thermostability improvements. This study provides guidance for enhancing thermostability of similar fold-type enzymes without impairing enzymatic activity in an efficient manner.