Obtaining Optical Purity for Product Diols in Enzyme-Catalyzed Epoxide Hydrolysis: Contributions from Changes in both Enantio- and Regioselectivity

Abstract

Enzyme variants of the plant epoxide hydrolase StEH1 displaying improved stereoselectivities in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene were generated by directed evolution. The evolution was driven by iterative saturation mutagenesis in combination with enzyme activity screenings where product chirality was the decisive selection criterion. Analysis of the underlying causes of the increased diol product ratios revealed two major contributing factors: increased enantioselectivity for the corresponding epoxide enantiomer(s) and, in some cases, a concomitant change in regioselectivity in the catalyzed epoxide ring-opening half-reaction. Thus, variant enzymes that catalyzed the hydrolysis of racemic (2,3-epoxypropyl)benzene into the R-diol product in an enantioconvergent manner were isolated.

Laboratory‐Evolved Enzymes Provide Snapshots of the Development of Enantioconvergence in Enzyme‐Catalyzed Epoxide Hydrolysis

“…This binding mode involves π–π interactions between the epoxide phenyl ring and the side chains of F33 and F189. The involvement of F33 in substrate binding might explain the degree of conservation of this residue during the directed evolution (only exchanges to tyrosine were observed) 3. The docking also proposed previously neglected residues as putative targets for future mutagenesis studies (including F191 and F158 interacting with ( R )‐ 1 and Y183 and F301 interacting with ( S )‐ 1) .…”

“…3 [b] Calculated distance between D105 and C‐1/C‐2 of the epoxide ring of 1 . [c] Mode 1′ involves interaction of the phenyl ring and H300; Mode 2 directs the phenyl ring of the substrate towards W/L106; in Mode 3, the phenyl substituent is sandwiched between F189 and F33 (Figure 2).…”

“…The enrichment appears to be a consequence of a combination of changes in both enantio‐ and regiopreference (to different degrees in different enzyme variants). Interestingly, the regioselectivity for C‐2 in the hydrolysis of ( R )‐ 1 was preserved in all variants, but was shifted towards C‐1 with ( S )‐ 1 3. As epoxide ring opening at the asymmetric carbon C‐1 causes inversion of the product's stereoconfiguration, these variants also produced ( R )‐ 2 from ( S )‐ 1 .…”

“…Solanum tuberosum epoxide hydrolase 1 (StEH1) is an attractive target for protein engineering, as it has been shown to be susceptible to changes in environment and structure, both of which can affect the enantio‐ and regioselectivity of this enzyme towards a range of epoxide substrates 1, 2, 3. StEH1 catalyzes the hydrolysis of trans ‐ and monosubstituted epoxides by a two‐step mechanism 4.…”

“…We have obtained, by ISM‐driven5 directed evolution, enzyme variants that were able to catalyze the formation of the hydrolysis product ( 2 ) with enriched optical purity 2, 3. We also isolated laboratory‐evolved variants that catalyze the production of enriched ( R )‐ 2 from racemic 1 .…”