QM/MM MD Simulations on the Origin of Donor/Acceptor Selectivity of Family GH51 α-L-Arabinofuranosidase-Catalyzed Hydrolysis and Transglycosylation Reactions

α-l-Arabinofuranosidase (EC 3.2.1.55) plays a crucial role in the degradation and processing of arabinoxylan and arabinoxylo-oligosaccharides (AXOS), making it a promising target for industrial biomass conversion and biotechnological applications. Despite its ability to cleave α(1→2), α(1→3), and α(1→5) glycosidic bonds, the origin of its preference for specific AXOS linkages remains unclear. Using quantum mechanics (QM) calculations and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, we investigated the catalytic mechanism and selectivity of hydrolysis and transglycosylation in a GH51 α-l-arabinofuranosidase from <i>Thermobacillus xylanilyticus</i>. We assessed various synthetic substrates and mono- and disubstituted AXOS (<i>p</i>NP-Ara<i>f</i>, A³, A²X, A³X, XA²X, XA³X and XA²⁺³X), predicting their binding modes and substrate specificity. Glu176 (acid/base) and Glu298 (nucleophile) facilitate AXOS hydrolysis via a two-step, double-displacement mechanism, with the second step (deglycosylation) being the rate-limiting step. Based on combined QM/MM MD and QM(ONIOM) studies, His240 is most likely protonated to maintain the positioning of the Glu176 residue within the Asp297-His240-Glu176 catalytic triad, thereby enabling efficient catalysis via an optimal electrostatic environment. Consistent with the experiment, the enzyme efficiently hydrolyzes <i>p</i>NP-Ara<i>f</i> and A³, with free-energy barriers of 14.8-15.4 kcal mol^-1 (compared to 17.9-29.3 kcal mol^-1 for AXOS); it selectively cleaves Ara<i>f</i>-α(1→3) over Ara<i>f</i>-α(1→2) linkages of donor substrates and favors xylobiose as an acceptor over aryl α-d-xylopyranoside in transglycosylation due to lower reaction barriers and effective transition-state stabilization. Our study provides a theoretical foundation for the engineering of this GH51 enzyme for efficient hydrolysis and synthesis of valuable xylan-based products.