Alleviating Substrate Inhibition of Leucine Dehydrogenase by Enhancing NAD+ Dissociation Efficiency

Leucine dehydrogenase catalyzes the asymmetric reductive amination of ketoacids to produce valuable chiral unnatural amino acids, but its application is often constrained by substrate inhibition. Here we employed a rational engineering strategy to alleviate substrate inhibition by enhancing the dissociation efficiency of NAD⁺. Notably, the variant H184A exhibited a 1.53-fold increase in enzyme activity and a 58% improvement in the half-maximal inhibitory concentration for 2-oxobutyric acid, alleviating substrate inhibition.