Identification, Characterization, and Mechanism of the Ene Reductase KlebER3 from <i>Klebsiella</i> sp. O852 for the Production of Dihydrocarvone

Dihydrocarvone is extensively utilized in food, pharmaceutical, and cosmetic fields because of its pleasant odor, high economic value, and diverse pharmacological properties. In this study, an ene reductase KlebER3, which catalyzed the conversion of carvone to dihydrocarvone using NAD(P)H as a cofactor, was identified from Klebsiella sp. O852 through genome sequencing. This enzyme displayed optimal activity at 30 °C and pH 7 and maintained appreciable stability at 20–50 °C and pH 6–8. Besides, KlebER3 had good resistance to dimethyl sulfoxide and glycerine and exhibited wide-ranging substrate acceptance for α,β-unsaturated compounds. Furthermore, a Q26A mutant with enhanced activity, stability, and stereoselectivity was obtained by molecular docking, alanine scanning, and site-directed mutagenesis. Molecular dynamics simulations revealed that these improvements may be attributed to increased nonpolar interactions, elevated flexibility of the active pocket, reduced binding free energy, and strengthened rigidity of protein conformation. This study established a basis for further investigations of the function and modification of ene reductase as well as the efficient biosynthesis of dihydrocarvone.