Reshaping the Catalytic Pocket of Glucose Dehydrogenase Es GDH for Stereocomplementary Asymmetric Synthesis of Chiral Aryl Lactones

Abstract Enzymatic catalytic promiscuity offers an effective strategy to extend the synthetic scope of native enzymes toward non-natural transformations. Glucose dehydrogenases (GDHs) are extensively used for glucose oxidation and NAD(P)H regeneration, however, their intrinsic ketoreductase promiscuity remains largely underexplored. Herein, a GDH from Exiguobacterium sibiricum (EsGDH) was engineered into a stereocomplementary biocatalyst for the asymmetric reduction of γ-keto esters to valuable γ-aryl-γ-butyrolactones. Through structure-guided semi-rational design, two enantiocomplementary variants, M3S (H148T/E97W/N197M) and M4R (W153K/E97A/V210W/I196L), were constructed, showing 318.4-fold and 257.5-fold increase in activity over the wild-type (WT), respectively. M3S and M4R enabled efficient synthesis of both (S)- and (R)-lactones in up to >99% ee. Molecular dynamics (MD) simulations demonstrated that asymmetric remodeling of the catalytic pocket governs EsGDH's stereoselectivity. Moreover, we conducted gram-scale biocatalytic preparation of (S)-γ-phenyl-γ-butyrolactone ((S)-1b), which was used to develop the chemoenzymatic route for (R)-fluoxetine and (R)-atomoxetine, confirming the practical potential of the catalytic system in the green manufacturing of chiral pharmaceuticals. This work broadens the synthetic applicability of promiscuous GDHs and provides a general strategy for engineering stereocomplementary biocatalysts for asymmetric synthesis.