A Single Hydrogen Bond Controls the Selectivity of Transglycosylation vs Hydrolysis in Family 13 Glycoside Hydrolases

Converting glycoside hydrolases (GHs)
from hydrolytic to synthetic
enzymes via transglycosylation is a long-standing goal for the biosynthesis
of complex carbohydrates. However, the molecular determinants for
the selectivity of transglycosylation (T) vs hydrolysis (H) are still
not fully unraveled. Herein, we show experimentally that mutation
of one active site residue can switch the enzyme activity between
hydrolysis and transglycosylation in two highly homologous GHs. Further
QM/MM simulations reveal that the mutation modulates the T vs H reaction
barriers via the presence/absence of a single H-bond with the nucleophile
Asp. Such a H-bond controls the product selectivity via a dual effect:
on one hand, it facilitates the breaking of the glycosyl-enzyme intermediate.
On the other, it displaces the sugar acceptor, resulting in a reduced
affinity and significant steric repulsion for transglycosylation.
These findings expand our understanding of the molecular mechanisms
that modulate the T/H balance in GHs.