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

Abstract

Abstractl‐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.magnified image