A Rational Active-Site Redesign Converts a Decarboxylase into a C═C Hydratase: “Tethered Acetate” Supports Enantioselective Hydration of 4-Hydroxystyrenes

Abstract

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from <i>Enterobacter</i> sp. (FDC_<i>Es</i>) 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 (<i>S</i>)-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.