Counteracting the Activity‐Diastereoselectivity Trade‐Off of l‐Threonine Aldolase by Regulating the Proton Transfer Microenvironment

Abstract

l-threonine aldolase (LTA) is a vital tool for the production of β-hydroxy-α-amino acids, important pharmaceutical intermediates with two chiral centres. However, the trade-off between activity and diastereoselectivity seriously hinders the application of LTA. Here, microenvironment of the proton transfer was regulated to improve the enzyme activity while avoiding the loss of diastereoselectivity. A combinatorial active-site saturation test (CAST) strategy was applied to engineer the microenvironment of the key histidines H86 and H128 involved in proton transfer. Except for the amino acid residues tunning diastereoselectivity, a total of 18 (9 + 9) residues lining around H86 and H128 were investigated. As a result, two variants, RS1-T92V and RS1-E123R, were obtained with specific activity from 9.61 U/mg to 11.24 U/mg and 14.41 U/mg, respectively. By combinatorial mutagenesis, a double-point mutant RS1-VR (T92V/E123R) was obtained with specific activity reaching 18.65 U/mg that was two-fold of the original strain (RS1). Notably, the mutant RS1-VR remained a high de value of 94.21%. Molecular dynamics (MD) simulations provided insights into the mechanism of activity-diastereoselectivity trade-off. The improvement of microenvironment contributes to reduce the swing amplitude of the side chain of H86, resulting in the proton transfer more efficient. This work provides a strategy of regulating the proton transfer microenvironment for counteracting the trade-off between activity and diastereoselectivity in protein engineering.