Reshaping Substrate-Binding Pocket of Leucine Dehydrogenase for Bidirectionally Accessing Structurally Diverse Substrates

Steric hindrance modification-based pocket reshaping is an effective approach for enzyme–substrate acceptance evolution. However, previous studies were limited to using the unidirectional acceptance trait of bulky substrates as a measure of fitness. In this endeavor, we conducted steric hindrance modification-based bidirectional pocket reshaping of Exiguobacterium sibiricum leucine dehydrogenase (EsLeuDH) for elucidating the differences in the molecular mechanism of pocket steric hindrance on the adaptability between the bulky and small substrates. A site-directed mutagenesis library generated based on the specifically chosen bidirectional mutagenesis sites and building blocks enabled the substrate specificity of EsLeuDH to be extended to both the small aliphatic 1a and bulky aromatic 1h; the catalytic efficiency toward 1b–g increased by 2.5–16.3-fold. Kinetic parameter determination revealed that the increased acceptance of the mutants toward small and bulky substrates was attributed to the decreased Km and enhanced kcat values, respectively. Structure-based computational analysis provided insights into the increased acceptance in both the steric hindrance strengthening and attenuating directions, which was attributed to the reshaped pocket with a favorable attack distance and an expanded catalytical binding space, respectively. Our study elucidates the mechanism difference of pocket steric hindrance of EsLeuDH on the adaptability of different types of substrates based on the implementation of bidirectional pocket reshaping, with potential applications in the divergent substrate acceptance evolution of amino acid dehydrogenase family members and other oxidoreductases with analogous substrate-binding pocket.