Rational design of a zinc-dependence secondary alcohol dehydrogenase to boost its oxidation activity for...

α-Hydroxyketones, which have significant industrial applications, can be sustainably synthesized through the oxidation of secondary alcohols using secondary alcohol dehydrogenase (SADH). However, the activity of the SADH oxidation reaction is generally low, making it unsuitable for large-scale production. In this study, a rational design approach was employed to computationally engineer SADH derived from Ogataea parapolymorpha (OpSADH), significantly enhancing its oxidation activity towards (R)-1,2-propanediol (PDO). The mutant M2 (S222T/S316A) exhibited a 14.2-fold increase in specific enzyme activity compared to the wild type (WT) and was employed as a catalyst for high-concentration hydroxyacetone production, facilitated by an NAD⁺ regeneration system. By applying an appropriate Zn²⁺ ion force field in molecular dynamics (MD) simulations, it was found that two mutation sites could stabilize the conformations of NAD⁺ and PDO, thereby revealing the molecular mechanism behind the enhanced activity of this metalloenzyme mutant. Notably, we first uncovered the dynamic mechanism by which four key residues (Cys39, His75, Glu76, and Glu175) and PDO within the active pocket contribute to the formation of coordination bonds with Zn²⁺. The findings of this study provide robust support for researching the catalytic mechanisms and dynamics processes of metalloenzymes.