Molecular Basis for Altered Hydrolysis and Transglycosylation in a GH20 β- N -Acetylhexosaminidase Mutant: Insights from Molecular Dynamics Simulations

Abstract β-N-acetylhexosaminidase (NAHase) plays a critical role in the potential high-value transformation of chitin through its hydrolysis and transglycosylation activities. In this work, a novel GH20 NAHase (designated Fmhex) from Fulvivirga maritima was heterologously expressed and biochemically characterized, which showed the highest activity at 45 °C and pH 6.0. To regulate its catalytic properties, a saturated mutation library targeting five nonconserved residues was constructed. Mutants Fmhex(G417A) and Fmhex(I436S) demonstrated 2.3-fold hydrolysis activity and 1.9-fold transglycosylation activity, respectively. Classical molecular dynamics alone cannot fully explain the enhanced hydrolysis of Fmhex(G417A) since it only exhibited a shorter catalytic distance and increased sugar-ring distortion, without a complete near-attack geometry or binding advantage. Conversely, Fmhex(I436S) reshaped the internal pocket architecture, enabling more pocket contacts and hydrogen bonds, deeper burial, and more favorable energetics, providing a structural rationale for its enhanced transglycosylation. These findings provided mechanistic insights and testable hypotheses for engineering NAHases with tailored hydrolysis/transglycosylation balances.