Inspired by the conformational change of the enzyme–substrate
complex in molecular dynamics (MD) simulation with distance restriction,
we propose a strategy for identifying the engineering targets based
on the comparative analysis of enzyme-/substrate- binding modes in
MD simulations with and without distance restriction (prereaction-state
simulation and free-state simulation). Taking the short-chain dehydrogenase/reductase
(SDR) mutant EbSDR8-G94A/S153L (Mu0) with poor activity toward bulky
aryl ketones as an example, H145 and Y188 were identified as the engineering
targets due to the distinct conformation difference in the two simulation
modes. To break the “beam” structure formed by these
residues at the entry of cavity C2 in free-state simulation, the substrate-binding
pocket was reconstructed, and meanwhile the relative size of cavities
C1 and C2 was modulated to improve the enantioselectivity. In this
way, mutants for efficient asymmetric reduction of o-halogenated acetophenones,
propiophenones, aromatic ketoesters, and diaryl ketones were designed,
delivering chiral alcohols with >99% conversion and >98% ee.
The effectiveness of this design strategy was also validated by the
successful redesign of PpYSDR, generating a variant for efficient
reduction of (4-chlorophenyl) 2-pyridyl ketone into the S-product with >99% conversion and 96% ee. MD simulations suggested
a suitable binding pocket with proper energy contribution as the ubiquitous
mechanism for the improvement of activity and enantioselectivity toward
substrates with varied structures. The success in this study provides
hints for the rational design of alcohol dehydrogenases/reductases
with both a broad substrate spectrum and high enantioselectivity.
As a chiral precursor for the important anticoagulant Edoxaban, enantioselective synthesis of (S)-3-cyclohexene-1-carboxylic acid is of great significance. The complicated procedures and generation of massive solid waste discourage its chemical synthesis, and the alternative biocatalysis route calls for an enzyme capable of asymmetric hydrolysis of racemic methyl-3-cyclohexene-1-carboxylate. To this end, we engineered the E. coli esterase BioH for improved S-enantioselectivity via rational design. By combinatorial modulation of steric and aromatic interactions, a positive mutant Mu3 (L24A/W81A/L209A) with relatively high S-selectivity in hydrolyzing racemic methyl-3-cyclohexene-1-carboxylate was obtained, improving the enantiomeric excess from 32.3% (the wild type) to 70.9%. Molecular dynamics simulation was conducted for both (R)- or (S)- complexes of the wild type and Mu3 to provide hints for the mechanism behind the increased S-selectivity. Moreover, the reaction conditions of Mu3 in methyl-3-cyclohexene-1-carboxylate hydrolysis was optimized to improve the conversion rate to 2 folds.
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