l‐threonine aldolase (LTA) catalyzes C−C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active‐site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn. Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti. Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C−C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C−C bond‐forming enzymes.

The L-threonine aldolase from Leishmania major was engineered to improve diastereoselectivity by a CAST/ISM strategy, providing insights into the relationship between physico -chemical properties of substrate access path and diastereoselectivity....

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.

L-threonine aldolase (LTA) catalyzes CÀ C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/ V143R was improved from 37.2 % syn to 99.4 % syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/ W307E was inverted to 97.2 % anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the CÀ C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other CÀ C bond-forming enzymes.