Potato
epoxide hydrolase 1 exhibits rich enantio- and regioselectivity
in the hydrolysis of a broad range of substrates. The enzyme can be
engineered to increase the yield of optically pure products as a result
of changes in both enantio- and regioselectivity. It is thus highly
attractive in biocatalysis, particularly for the generation of enantiopure
fine chemicals and pharmaceuticals. The present work aims to establish
the principles underlying the activity and selectivity of the enzyme
through a combined computational, structural, and kinetic study using
the substrate trans-stilbene oxide as a model system.
Extensive empirical valence bond simulations have been performed on
the wild-type enzyme together with several experimentally characterized
mutants. We are able to computationally reproduce the differences
between the activities of different stereoisomers of the substrate
and the effects of mutations of several active-site residues. In addition,
our results indicate the involvement of a previously neglected residue,
H104, which is electrostatically linked to the general base H300.
We find that this residue, which is highly conserved in epoxide hydrolases
and related hydrolytic enzymes, needs to be in its protonated form
in order to provide charge balance in an otherwise negatively charged
active site. Our data show that unless the active-site charge balance
is correctly treated in simulations, it is not possible to generate
a physically meaningful model for the enzyme that can accurately reproduce
activity and selectivity trends. We also expand our understanding
of other catalytic residues, demonstrating in particular the role
of a noncanonical residue, E35, as a “backup base” in
the absence of H300. Our results provide a detailed view of the main
factors driving catalysis and regioselectivity in this enzyme and
identify targets for subsequent enzyme design efforts.
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.
Computational studies highlight the importance of conformational diversity in the enantioconvergent hydrolysis of styrene oxide by potato epoxide hydrolase 1.
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.
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