Abstract: (S)-3-Hydroxytetrahydrofuran is an important pharmaceutical intermediate, and its chemical synthesis meets challenges such as low stereoselectivity and high cost. Alternatively, alcohol dehydrogenases catalyze the one-step asymmetric reduction of dihydro-3(2H)-furanone to the corresponding alcohol product. In this study, after screening an alcohol dehydrogenase library, an alcohol dehydrogenase derived fromAromatoleum aromaticumEbN1 (PED) was obtained, which enabled the reduction of dihydro-3(2H)-furanone to (S)-3-hydroxytetrahydrofuran. By optimizing the reaction conditions, PED catalyzed the model reaction with a >99% conversion. The catalytic efficiency (kcat/Km) was measured at 0.40 L/（mmol·s）, with aneevalue of 90%. Additionally, PED demonstrated a good thermostability, maintaining aTmvalue of 67 ℃. Computational analysis revealed the mechanism of stereoselectivity in the reduction of substrates catalyzed by PED. The key residues such as S141, Y151 and Y154 formed a network of hydrogen-bonding interactions with the substrate, which promoted the formation of apro-(S) conformation of the substrate. The (S)-product was then formed by the migration of a hydride from the coenzyme NADH to the carbonyl carbon of the substrate. Furthermore, large-scale reactions showed that PED can completely convert the substrate concentration up to 400 mmol/L in 5 h, with an isolated yield of 74.7% and aneevalue of 90%. In this study, a novel alcohol dehydrogenase PED was identified and characterized with enzymatic performance, expanding the repertoire of enzymes available for the biocatalysis of (S)-3-hydroxytetrahydrofuran.

Key words: alcohol dehydrogenase, biocatalysis, asymmetric reduction, (S)-3-hydroxytetrahydrofuran, cofactor regeneration