Improved enantioselectivity of E. coli BioH in kinetic resolution of methyl (S)-3-cyclohexene-1-carboxylate by combinatorial modulation of steric and aromatic interactions

Abstract

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.

Rational Design of Dehydrogenase/Reductases Based on Comparative Structural Analysis of Prereaction-State and Free-State Simulations for Efficient Asymmetric Reduction of Bulky Aryl Ketones

“…Although the active site residue Y156 offered higher energy contribution in Mu14-6a ProS , S143 made no contribution to the binding of 6a ProS . Moreover, K160 showed a positive value of energy decomposition with 0.96 (12) kcal/mol of energy decomposition to Mu14-6a ProS , which is unfavorable for accomplishing the function of catalytic residues and cofactor. On the contrary, the active sites S143, Y156, and K160 all made contribution to Mu14-6a ProR with −0.30(9), −0.67 (13), and −0.18 (9) kcal/mol of energy decomposition, respectively, which is favorable for realizing proton transfer and electron transfer.…”

“…Protein engineering has demonstrated its power in improving the catalytic performance of enzymes. − For the mutants generated by protein engineering, computational molecular dynamics simulation has been widely employed to interpret the mechanism responsible for the change in enzyme features such as activity, stability, and enantioselectivity. , The emergence of constrained MD simulation made the analysis of the prereaction state possible, and since then, the difference in formation probability and stability of the prereaction state was used to explain the activity difference in various reaction systems. − Constrained MD simulation applies an external force onto a physical system, which drives a change in coordinates. , Hence, we put forward a hypothesis: the binding mode of the substrate/enzyme complex in prereaction-state simulation (with distance restriction, defined as T-state simulation) would be distinctly different from that in free-state simulation (without distance restriction, defined as F-state simulation) if the binding pocket is not suitable for accommodating the target substrate. Based on this hypothesis, a strategy is proposed to identify the engineering targets for enzyme redesign toward unnatural substrates by searching for the key residues with obviously different conformations in T-state simulation and F-state simulation.…”

Engineered Cyclohexylamine Oxidase with Improved Activity and Stereoselectivity for Asymmetric Synthesis of a Bulky Dextromethorphan Precursor and Its Analogues

“…Recently, comparative analysis of enzyme-/substrate-binding modes in molecular dynamics (MD) simulations with and without distance restriction, namely prereaction-state simulation (defined as T-state simulation) and free-state simulation (defined as F-state simulation), has been demonstrated as a useful strategy both to rationalize the observed enzyme activity as well as stereoselectivity, and to help efficiently identify potential amino acid targets for protein engineering. [54][55][56][57][58] In the present study, this strategy was invoked to gain insight into the molecular basis of the improved activity and stereoselectivity of the evolved mutant WXF-FM. Accordingly, a 3-D structure of this enzyme variant with five-point mutations was first built with SWISS-MODEL server [59] by using the ternary crystal structure (PDB ID: 6LQL) of wild-type CHAO CCH12-C2 as the template.…”

Kinetic Resolution of Nearly Symmetric 3-Cyclohexene-1-carboxylate Esters Using a Bacterial Carboxylesterase Identified by Genome Mining

“…However, commercial enzymes are generally expensive, and the reproducibility of their outcomes depends on their commercial availability. Recently, the carboxylesterase BioH, which is involved in biotin synthesis in Escherichia coli , was reported to hydrolyze rac -CHCM; however, it had an ee of only 32.3% and an enantioselectivity value ( E value) of 2.1 . Through the combinatorial modulation of steric and aromatic interactions, the best-performing variant of BioH, Mu3 (L24A/W81A/L209A), reached a 70.9% ee and an E value of 7.1 at 40 mM rac -CHCM.…”

“…Furthermore, the space-time yields of the commercial enzymes are lower than 20 g·L –1 ·d –1 (Table S3). The variant Mu3 of BioH was previously constructed to catalyze the kinetic resolution of rac -CHCM. , However, the substrate loads are relatively low (<10 g·L –1 ), and the S/C ratio of 2 g·g –1 and the low space-time yield are not acceptable for industrial applications. Among biocatalysts capable of the enantioselective hydrolysis of rac -CHCM to ( S )-CHCM, CarEst3 identified in this study exhibits the highest substrate tolerance (700 g·L –1 ), S/C value (3500 g·g –1 ), and space-time yield (538 g·L –1 ·d –1 ), demonstrating its great potential in industrial manufacturing.…”