Unlocking Loop Geometric Remodeling Redirects Aldo-Keto Reductase from Substrate Promiscuity to 3-Keto-Deoxynivalenol Specificity

Aldo-keto reductase is responsible for the formation of nontoxic 3-<i>epi</i>-deoxynivalenol (3-<i>epi</i>-DON) from 3-keto-DON, which is an enzymatic detoxification manner to completely eliminate the toxicity of mycotoxin DON against health and environmental threats. Therefore, unlocking the facilitated substrate specificity of aldo-keto reductase for 3-keto-DON has become a critical challenge for advanced catalytic performance. In this endeavor, a loop-based engineering strategy was developed for aldo-keto reductase AKR13B3 from <i>Devosia</i> A6-243 to catalyze 3-keto-DON. A 31.9-fold switch from a disfavored substrate to the preferred one was produced along with a significant 37.9-fold increase in catalytic efficiency. Kinetic parameter determinations, structural analyses, and molecular simulations were employed to elucidate the molecular mechanisms underlying these enhancements in catalytic activity and substrate specificity. Overall, our work presents a feasible scheme for designing aldo-keto reductases with exceptional substrate specificity and catalytic activity, holding great promise for developing enzymatic detoxification agents.