Semirational engineering of an aldo–keto reductase KmAKR for overcoming trade‐offs between catalytic activity and thermostability

Abstract

Enzyme engineering usually generates trade-offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo-keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity.Previously, we constructed KmAKR M9 (W297H/Y296W/K29H/Y28A/T63M/A30P/ T302S/N109K/S196C), which showed outstanding activity towards t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), and t-butyl 6-cyano-(3R,5R)dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin.Under the guidance of computer-aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKR M13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/ K164E/S232A/S182H/Q266D). The T m and T 50 15 values of KmAKR M13 were 10.4 and 6.1°C higher than that of KmAKR M9 , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKR M9 . Structural analysis indicated that stabilization of the α-helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKR M13 completely asymmetrically reduced 400 g/l t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)-CDHH (>99.5% d.e. P ) with a space-time yield (STY) of 449.2 g/l•d.

Enhancing the organic solvent resistance of transaminase from Aspergillus terreus by regional random mutation

“…However, in many other cases of trade-off escape between activity and stability, the location of beneficial substitution is far from enzyme active site. [31][32][33] The beneficial residue position 23, 200, and 260 in At ATA with improving the organic solvents stability, locate at loop region of surface that are far from active center, which may explain why M3 enhanced organic solvent stability without sacrificing activity.…”

Enabling biocatalysis in high‐concentration organic cosolvent by enzyme gate engineering

“…It is interesting that the M2 F56V and M2 F56D exhibited both improved activity and DMSO resistance (escaped the “activity–stability trade‐off”), whereas the variants M2 F56H and M2 F56G displayed increased activity but lower DMSO resistance. Previous reports show that both the location and type of a mutated residue affect the enzymatic properties trade‐off (Li et al, 2021; Tokuriki et al, 2008). The “activity–stability trade‐off” often appeares in the variants harboring the beneficial substitutions located at or near the active site.…”

Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design

“…28 Many vivid examples have demonstrated its effectiveness not only in tailoring the catalytic activity, but also in improving the thermostability of enzymes. [32][33][34][35] To predict residues that might affect the enzyme's thermostability, a 3D homology models of LrLDH was constructed (Fig. 1a) and uploaded to the HotSpot Wizard web server.…”