The catalytic promiscuity of a ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across the C=C bond of hydroxystyrenes was extended to the N‐, C‐ and S‐nucleophiles methoxyamine, cyanide and propanethiol to furnish the corresponding addition products in up to 91% ee. The products obtained from the biotransformation employing the most suitable enzyme/nucleophile pairs were isolated and characterized after optimizing the reaction conditions. Finally, a mechanistic rationale supported by quantum mechanical calculations for the highly (S)‐selective addition of cyanide is proposed.
The
promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes
catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the
active site, which serves as a proton relay activating a water molecule
for nucleophilic attack on a quinone methide electrophile. This “cofactor”
is crucial for achieving improved conversions and high stereoselectivities
for (S)-configured benzylic alcohol products. Similar
effects were observed with simple aliphatic carboxylic acids as additives.
A rational redesign of the active site by replacing the bicarbonate
or acetate “cofactor” with a newly introduced side-chain
carboxylate from an adjacent amino acid yielded mutants that efficiently
acted as C=C hydratases. A single-point mutation of valine
46 to glutamate or aspartate improved the hydration activity by 40%
and boosted the stereoselectivity 39-fold in the absence of bicarbonate
or acetate.
The binding of two photoswitches to the human voltage-gated sodium channel NaV1.4 was computationally investigated, and the main interactions involved in the process were unveiled and characterized.
The operability and
substrate scope of a redesigned vinylphenol
hydratase as a single biocatalyst or as part of multienzyme cascades
using either substituted coumaric acids or phenols as stable, cheap,
and readily available substrates are reported.
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