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

Abstract

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 K m and enhanced k cat 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.

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production

“…42 The appropriate pocket size is necessary for an enzyme to interact efficiently with ligands. 26 As shown in Figure 5A,B, the distance between residues S267 and S180 (d (S180 − S267) ), on either side of the BuLTTA active site pocket, was used as an indicator to evaluate changes in the active site pocket. The value of d (S180 − S267) was 1 Å shorter in mutant M4 than in the WT enzyme, suggesting that the active site pocket of M4 was smaller (fine-tuned and more conducive to stable ligand binding).…”

“…25 Wu et al successfully reshaped the active site pocket of isoleucine dehydrogenase from Exiguobacterium sibiricum by analyzing its sequence conservation and the pocket size. 26 To date, engineering of LTTAs has mainly focused on directed evolution. 27 However, even with efficient high-throughput screening methods, the effort required is still relatively high (typically screening thousands of variants) compared to rational design.…”

“…According to the sequence–structure–function relationship, rational design can greatly reduce labor intensity. − Liu et al rationally replaced I258 with M258 in the active site of 7α-hydroxysteroid dehydrogenase from Brucella melitensis, which significantly improved the catalytic efficiency and thermal stability of the enzyme . Wu et al successfully reshaped the active site pocket of isoleucine dehydrogenase from Exiguobacterium sibiricum by analyzing its sequence conservation and the pocket size . To date, engineering of LTTAs has mainly focused on directed evolution .…”

Rational design of enzyme activity and enantioselectivity

“…Several studies have reported the successful application of steric hindrance based rational design strategy in enzyme activity enhancement towards bulky substrates ( Li F. et al, 2022 ; Wang Z. et al, 2022 ; Wu et al, 2022 ). α-ketoglutarate (α-KG) is the natural substrate of glutamate dehydrogenase (GluDH).…”

Enhancement of the substrate specificity of D‐amino acid oxidase based on tunnel‐pocket engineering

“…The design engineering of enzymes is a valuable tool for enhancing biocatalytic diversity. One method of expanding the enzyme substrate scope is by reshaping the substrate‐binding pocket (Sacchi et al, 2002; Wu et al, 2022). The usual case is saturation mutagenesis of residues at a certain distance from the substrate, which will not change the reaction mechanism of the enzyme.…”