Enantioconvergent hydrolysis by epoxide hydrolase is a promising method for the synthesis of important vicinal diols. However, the poor regioselectivity of the naturally occurring enzymes results in low enantioconvergence in the enzymatic hydrolysis of styrene oxides. Herein, modulated residue No. 263 was redesigned based on structural information and a smart variant library was constructed by site-directed modification using an "optimized amino acid alphabet' to improve the regioselectivity of epoxide hydrolase from Vigna radiata (VrEH2). The regioselectivity coefficient (r) of variant M263Q for the R-isomer of meta-substituted styrene oxides was improved 40-63-fold, and variant M263V also exhibited higher regioselectivity towards the R-isomer of para-substituted styrene oxides compared with the wild type, which resulted in improved enantioconvergence in hydrolysis of styrene oxide scaffolds. Structural insight showed the crucial role of residue No. 263 in modulating the substrate binding conformation by altering the binding surroundings. Furthermore, increased differences in the attacking distance between nucleophilic residue Asp101 and the two carbon atoms of the epoxide ring provided evidence for improved regioselectivity. Several high-value vicinal diols were readily synthesized (>88% yield, 90%-98% ee) by enantioconvergent hydrolysis using the reprogrammed variants. These findings provide a successful strategy for enhancing the enantioconvergence of native epoxide hydrolases through key single-site mutation and more powerful enzyme tools for the enantioconvergent hydrolysis of styrene oxide scaffolds into single (R)-enantiomers of chiral vicinal diols.
Title: A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis Zheng YC, Ding LY, Jia Q, Lin Z, Hong R, Yu HL, Xu JH Ref: Chembiochem, 22:996, 2021 : PubMed
Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.
Title: Electrostatic Effect of Functional Surfaces on the Activity of Adsorbed Enzymes: Simulations and Experiments Zheng H, Yang SJ, Zheng YC, Cui Y, Zhang Z, Zhong JY, Zhou J Ref: ACS Appl Mater Interfaces, 12:35676, 2020 : PubMed
The efficient immobilization of haloalkane dehalogenase (DhaA) on carriers with retaining of its catalytic activity is essential for its application in environmental remediation. In this work, adsorption orientation and conformation of DhaA on different functional surfaces were investigated by computer simulations; meanwhile, the mechanism of varying the catalytic activity was also probed. The corresponding experiments were then carried out to verify the simulation results. (The simulations of DhaA on SAMs provided parallel insights into DhaA adsorption in carriers. Then, the theory-guided experiments were carried out to screen the best surface functional groups for DhaA immobilization.) The electrostatic interaction was considered as the main impact factor for the regulation of enzyme orientation, conformation, and enzyme bioactivity during DhaA adsorption. The synergy of overall conformation, enzyme substrate tunnel structural parameters, and distance between catalytic active sites and surfaces codetermined the catalytic activity of DhaA. Specifically, it was found that the positively charged surface with suitable surface charge density was helpful for the adsorption of DhaA and retaining its conformation and catalytic activity and was favorable for higher enzymatic catalysis efficiency in haloalkane decomposition and environmental remediation. The neutral, negatively charged surfaces and positively charged surfaces with high surface charge density always caused relatively larger DhaA conformation change and decreased catalytic activity. This study develops a strategy using a combination of simulation and experiment, which can be essential for guiding the rational design of the functionalization of carriers for enzyme adsorption, and provides a practical tool to rationally screen functional groups for the optimization of adsorbed enzyme functions on carriers. More importantly, the strategy is general and can be applied to control behaviors of different enzymes on functional carrier materials.
An epoxide hydrolase from Vigna radiata (VrEH2) affords partial enantioconvergence (84% ee) in the enzymatic hydrolysis of racemic p-nitrostyrene oxide (pNSO), mainly due to insufficient regioselectivity for the (S)-enantiomer (rS = alphaS/betaS = 7.3). To improve the (S)-pNSO regioselectivity, a small but smart library of VrEH2 mutants was constructed by substituting each of four key residues lining the substrate binding site with a simplified amino acid alphabet of Val, Asn, Phe, and Trp. Among the mutants, M263N attacked almost exclusively at Calpha in the (S)-epoxide ring with satisfactory regioselectivity (rS = 99.0), without compromising the original high regioselectivity for the (R)-epoxide (rR = 99.0), resulting in near-perfect enantioconvergence (>99% analytical yield, 98% ee). Structural and conformational analysis showed that the introduced Asn263 formed additional hydrogen bonds with the nitro group in substrate, causing a shift in the substrate binding pose. This shift increased the difference in attacking distances between Calpha and Cbeta, leading to an improved regiopreference toward (S)-pNSO and affording near-perfect enantioconvergence.
