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

Abstract

Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)‐3‐phenylpropane‐1,2‐diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)‐diol is not only a consequence of changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)‐benzyloxirane. In order to probe the structural origin of these differences in substrate selectivity and catalytic regiopreference, we solved the crystal structures for the evolved StEH1 variants. We used these structures as a starting point for molecular docking studies of the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking analysis, the apparent preferred binding modes appear to rationalize the experimentally determined regioselectivities. The analysis also identifies an active site residue (F33) as a potentially important interaction partner, a role that could explain the high conservation of this residue during evolution. Overall, our experimental, structural, and computational studies provide snapshots into the evolution of enantioconvergence in StEH1‐catalyzed epoxide hydrolysis.

Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking

“…An epoxide hydrolase from Solanum tuberosum (StEH1) is a well-studied enzyme with a buried active site and a well-defined main tunnel providing access into the catalytic triad. As an attractive target for protein engineering, it has been intensively studied using both experimental [16,17,18,19,20,21,22,23] and computational methods [18,20,23,24,25,26,27]. Solanum tuberosum epoxide hydrolase belongs to the α/β-hydrolase family.…”

“…The analysis of StEH1 enantioselectivity was performed in the series of studies [21,22,26,29]. Four hotspots were targeted by random mutagenesis that consisted of: A single F33 residue (hotspot A), the Y106 and L109 residues (hotspot B), V141, L145, and I155 residues (hotspot C) and I180 and F189 residues (hotspot D) [21].…”

“…Four hotspots were targeted by random mutagenesis that consisted of: A single F33 residue (hotspot A), the Y106 and L109 residues (hotspot B), V141, L145, and I155 residues (hotspot C) and I180 and F189 residues (hotspot D) [21]. Several constructed mutants were investigated in details [21,22] and the crystal structures were solved for four of them: R-C1 variant (V141K, I155V), R-C1B1 variant (W106L, L109Y, V141K, I155V), R-C1B1D33 variant (W106L, L109Y, V141K, I155V, F189L), and R-C1B1D33E6 variant (W106L, L109Y, V141K, I155V, F189L, L266G) [26,29]. All the targeted residues were situated in the binding site cavity, apart from F189, which was located behind the active site cavity.…”

“…The R-C1B1 variant was studied by Bauer et al [29], who pointed out that the origin of the observed enantio- and regioselectivity is related not only to active site cavity enlargement but also to water penetration into the active site. Analysis of the F189L variant [26] also showed the importance of the F33 residue, which was found to be involved in substrate binding and might explain the degree of conservation of this residue during directed evolution (only exchanges to tyrosine were observed) [22].…”

Epoxide hydrolysis as a model system for understanding flux through a branched reaction scheme

“…The experimentally determined rates could thus complement theoretical calculations and protein structure analyses. StEH1 is also unusually well suited as a model enzyme system for clarifying some fundamental aspects of enzyme catalysis and substrate recognition because (i) the chemical mechanism is well understood and its generality increases the scope of possible applicable related systems, as highly similar catalytic mechanisms are found in a wide range of hydrolytic enzymes, representing both related (/-hydrolases) as well as more structurally unrelated systems (for example enzymes such as the pancreatic proteases and subtilisin); (ii) the different alternative mechanistic possibilities for epoxide ring opening provide opportunities for explaining enantioselectivity and regioselectivity in the transformation of asymmetric substrates and, finally; (iii) crystal structures have been solved for all of the isolated variants (Mowbray et al, 2006;Bauer et al, 2016;Janfalk Carlsson et al, 2016). To further rationalize the functional data, we have performed detailed EVB calculations on the hydrolysis reaction catalyzed by both the wild-type enzyme and the variants studied here.…”