Reshaping the Binding Pocket of Aldo–Keto Reductase for Enhanced Stereoselectivity and Activity

The bioremediation strategy is a promising environmentally friendly alternative to eliminate deoxynivalenol (DON) contamination in feed and foodstuffs, but few direct and effective enzyme-catalyzed detoxification methods are currently available. Here, we identified an aldo-keto reductase with DON-degrading ability from the <i>Devosia</i> strain A6-243 (AKR6D2) through a stepwise gene mining approach. AKR6D2 was found to have great thermostability toward the reduction of C3 carbonyl oxygen of 3-keto- deoxynivalenol (3-keto-DON) but with poor stereoselectivity (44.97% <i>e.e.</i> for 3-<i>epi</i>- deoxynivalenol (3-<i>epi</i>-DON)), which limited its practical application. By reshaping the substrate-binding pocket of AKR6D2, a mutant W21A/G53N with excellent catalytic activity (330-fold higher) and stereoselectivity (>99% <i>e.e.</i> for 3-<i>epi</i>-DON) was obtained. Based on the insights provided by molecular dynamics simulations, W21A/G53N-3-keto-DON_proR was more favorable in the formation of prereaction states to produce the <i>R</i>-configuration product. This work confirmed that engineered AKR6D2 is a powerful biocatalyst for the detoxification of DON in contaminated food and feed.