Regioselectivity Engineering of Epoxide Hydrolase: Near-Perfect Enantioconvergence through a Single Site Mutation

Abstract

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 (r S = α S /β S = 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 Cα in the (S)-epoxide ring with satisfactory regioselectivity (r S = 99.0), without compromising the original high regioselectivity for the (R)-epoxide (r R = 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 Cα and Cβ, leading to an improved regiopreference toward (S)-pNSO and affording near-perfect enantioconvergence.

Reprogramming Epoxide Hydrolase to Improve Enantioconvergence in Hydrolysis of Styrene Oxide Scaffolds

“…asc.wiley-vch.de the molar ratio of diol isomers obtained using an optically pure substrate. [10] As shown in Table S1, both WT and M263V showed high r S values of more than 49.0 for the S-enantiomer of para-substituted substrates 1 a, 3 a, and 4 a, while the r R values of the wildtype for 1 a, 3 a, and 4 a were only 5.6, 4.3, and 3.5, respectively. Therefore, the insufficient regioselectivity of WT toward R-enantiomers 1 a, 3 a, and 4 a was the main origin of low enantioconvergence.…”

“…asc.wiley-vch.de pose that led to improved regioselectivity. [10] In contrast, mutating to Phe or Trp with bulky sidechains caused a severe decrease in regioselectivity. Herein, to rationally and efficiently construct variants for improved enantioconvergence towards other styrene oxide scaffolds, an "optimized amino acid alphabet" consisting of six pre-selected residues (Ala-Val-Ser-Cys-Asn-Gln) was designed in accordance with CAST (Combinatorial Active-site Saturation Test) method and the previous studies.…”

“…Subsequently, a semirational design strategy was adopted that revealed a crucial residue (Met263) for precise modulation of the regioselectivity. [10] This resulted in near-perfect enantioconvergence toward rac-pNSO after single-site mutation (M263N). However, the enantioconvergence of VrEH 2 must be improved for the enzymatic hydrolysis of other styrene oxides with different substitution groups.…”

One Pot Asymmetric Synthesis of (R)‐Phenylglycinol from Racemic Styrene Oxide via Cascade Biocatalysis

“…The genes of VrEH2 M263V and EaGDH were cloned in plasmid pET-28a (+), respectively, and the competent cells of E. coli BL21 (DE3) were then transformed with the resultant recombinant plasmids as described previously. [15,16] The gene of AlaDH was amplified by PCR from the genome DNA of Bacillus megaterium ECU1001 with the primers reported for alanine dehydrogenase from Bacillus megaterium WSH-002 and cloned in the plasmid pET-28a (+), and competent cells of E. coli BL21 (DE3) was then transformed with the recombinant plasmid. [22] The gene of ω-TA Y150F/V153A was cloned in plasmid pET-24b (+) and transformed into E. coli BL21 (DE3) competent cells as described in literature.…”

P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules

“…To alleviate this shortcoming, a range of base-modified decoy molecules was suggested. In essence, these dual functional small molecules (DFSMs) comprise an imidazole-base coupled via a linker moiety to an amino acid anchoring part in order to position the base within the P450 monooxygenases' active sites, thereby enabling peroxygenase-like reactions [18,19]. In the current study, we set out to validate and broaden this very interesting concept.…”