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

ABSTRACT 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.