Mechanistic Insights into How the Protonation State of D234 Dictates the Reactivity in Streptomyces coelicolor beta-N-Acetylhexosaminidase

beta-N-Acetylhexosaminidases (HEXs)play importantroles in human diseases and the biosynthesis of human milk oligosaccharides.Despite extensive research, the catalytic mechanism of these enzymesremains largely unexplored. In this study, we employed quantum mechanics/molecularmechanics metadynamics to investigate the molecular mechanism of Streptomyces coelicolor HEX (ScHEX),which has shed light on the transition state structures and conformationalpathways of this enzyme. Our simulations revealed that Asp242, locatednear the assisting residue, can switch the reaction intermediate toan oxazolinium ion or a neutral oxazoline, depending on the protonationstate of the residue. Moreover, our findings indicated that the freeenergy barrier of the second-step reaction starting from the neutraloxazoline increases steeply due to the reduction in the anomeric carbonpositive charge and the shortening of the C1-O-2N bond. Our results provide valuable insights into the mechanism ofsubstrate-assisted catalysis and could facilitate the design of inhibitorsand the engineering of analogous glycosidases for biosynthesis.