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

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

Development and Scaling-Up of the Fragrance Compound 4‑Ethylguaiacol Synthesis via a Two-Step Chemo-Enzymatic Reaction Sequence

The transformation of (abundant) oxygenated biomass-derived building blocks via chemo-enzymatic methods is a valuable concept for accessing useful compounds, as it combines the high selectivity of enzymes and the versatility of chemical catalysts. In this work, we demonstrate a straightforward combination of a phenolic acid decarboxylase (PAD) and palladium on charcoal (Pd/C) that affords the flavor compound 4-ethylguaiacol from ferulic acid. The use of a two-phase system proved to be advantageous in terms of enzyme activity, stability, and volumetric productivity and allows us to carry out the hydrogenation step directly in the organic layer containing exclusively the intermediate, vinylguaiacol. The enzymatic decarboxylation step in the biphasic system afforded 89% conversion of 100 mM (19 g L^–1) ferulic acid with an isolated yield of 75%. By extracting 4-vinylguaiacol continuously into the organic phase, conversion was enhanced to 92% using 170 mM (33 g L^–1) ferulic acid, which was only possible in the continuous extraction and distillation setup developed. The reaction cascade (PAD–Pd/C) is demonstrated at gram scale, affording the target product 4-ethylguaiacol (1.1 g) in 70% isolated yield in a two-step two-pot process. The enzymatic step was characterized in detail to overcome major constraints, and the process favorably compares in terms of the environmental impact with traditional approaches

Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives

The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed <i>o</i>-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the β-carbon atom of styrenes forming (<i>E</i>)-cinnamic acids

Biocatalytic Characterization of Human FMO5: Unearthing Baeyer–Villiger Reactions in Humans

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical <i>in vitro</i> activity of human FMO5 as a Baeyer–Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer–Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism

A Stereoselective Inverting <i>sec</i>-Alkylsulfatase for the Deracemization of <i>sec</i>-Alcohols

A metallo-β-lactamase-type alkylsulfatase was found to catalyze the enantioselective hydrolysis of <i>sec</i>-alkylsulfates with strict inversion of configuration. This catalytic event, which does not have an analog in chemocatalysis, yields homochiral (<i>S</i>)-configurated alcohols and nonreacted sulfate esters. The latter could be converted into (<i>S</i>)-<i>sec</i>-alcohols as the sole product in up to >99% ee via a chemoenzymatic deracemization protocol on a preparative scale