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

Åsa Janfalk Carlsson,Shina C L Kamerlin,Mikael Widersten,Paul Bauer,Doreen Dobritzsch

doi:10.1107/s2052252518003573

Abstract

The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl-propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure-activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

Highlights

Epoxides are important components in the toolbox of chiral building blocks for asymmetric synthesis (Santaniello et al, 1992)
As empirical valence-bond (EVB) is a parameterized approach, the quality of the results provided by this approach depends on two things: (i) the quality of the parameterization used in the work and (ii) the amount of conformational sampling performed and whether this is sufficient to obtain convergent results
The EVB simulations predict this low degree of regioselectivity with (R,R)-1a, with very similar barriers for hydrolysis of either the (R,S)- or (S,R)-alkylenzymes (C1 or C2; mode 1 in Table 3 and Figs. 7a and 8a)

Summary

Introduction

Epoxides are important components in the toolbox of chiral building blocks for asymmetric synthesis (Santaniello et al, 1992). The hydrolysis of trans-methylstyrene oxide (1a; Fig. 1a) is expected to occur via breakage of the bond between the benzylic C atom (C1) and the epoxide O atom This prediction is based on electron donation from the phenyl substituent, which stabilizes the buildup of positive charge on the attacked C atom (Fig. 1b; Parker & Isaacs, 1959). The reaction is further facilitated by protonation of the leaving-group oxide Making such regioselectivity predictions is far less trivial in an enzyme-catalyzed epoxide ring-opening reaction, since the chiral nature of the active site and its particular microenvironment steer the reaction in directions that are uncoupled from the intrinsic chemical reactivities (Monterde et al, 2004) of the epoxide substrates. To correctly predict the resulting stereoselectivity becomes a major and often unmet challenge in itself

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Conclusion