Calcium enhances hydrolytic and transfucosylation activities in α-L-fucosidase from Thermotoga maritima through catalytic loop stabilization: an MD simulation study

Abstract α-L-Fucosidases are versatile glycoside hydrolases capable of catalyzing both hydrolysis and transfucosylation reactions, making them valuable biocatalysts for the synthesis of fucosylated oligosaccharides (FucOS). However, transfucosylation efficiency is often limited by competing hydrolysis, and the factors governing this balance remain incompletely understood. In this study, we investigated the effect of divalent cations on the catalytic behavior of the α-L-fucosidase from Thermotoga maritima (FUC- Tm ) by combining enzymatic assays with molecular dynamics (MD) simulations and binding energy analyses. Among the tested ions, Ca 2+ produced the most pronounced effect, increasing both hydrolytic and transfucosylation activities, with the highest transfucosylation efficiency observed at intermediate CaCl 2 concentrations. MD simulations indicated that Ca 2+ does not induce major global conformational changes but is associated with reduced flexibility in loop regions surrounding the catalytic pocket, particularly Loop-B, and with improved positioning of catalytic residues. These effects are consistent with enhanced substrate interactions, as supported by binding energy analyses. However, the increase in transfucosylation efficiency coincided with a decrease in water activity, suggesting that the observed behavior arises from a combination of structural and physicochemical factors. Therefore, Ca 2+ -mediated modulation of FUC- Tm activity is best interpreted as a multifactorial process involving changes in local protein dynamics and in the reaction environment. These findings provide insight into ion-dependent modulation of glycoside hydrolases and highlight the importance of integrating structural dynamics and solvent effects to improve enzymatic synthesis of FucOS. Key points Calcium enhances both hydrolytic and transfucosylation activities of α-L-fucosidase from Thermotoga maritima under specific conditions. Molecular dynamics simulations indicate that Ca 2+ is associated with reduced flexibility in loop regions adjacent to the catalytic site, contributing to the maintenance of catalytic pocket geometry. The modulation of catalytic behavior is influenced by a combination of structural effects and physicochemical factors, including changes in water activity and ionic strength.