Conformational Switch of the 250s Loop Enables the Efficient Transglycosylation in GH Family 77

Amylomaltases (AMs) play important roles in glycogen
and maltose
metabolism. However, the molecular mechanism is elusive. Here, we
investigated the conformational dynamics of the 250s loop and catalytic
mechanism of Thermus aquaticus TaAM using path-metadynamics
and QM/MM MD simulations. The results demonstrate that the transition
of the 250s loop from an open to closed conformation promotes polysaccharide
sliding, leading to the ideal positioning of the acid/base. Furthermore,
the conformational dynamics can also modulate the selectivity of hydrolysis
and transglycosylation. The closed conformation of the 250s loop enables
the tight packing of the active site for transglycosylation, reducing
the energy penalty and efficiently preventing the penetration of water
into the active site. Conversely, the partially closed conformation
for hydrolysis results in a loosely packed active site, destabilizing
the transition state. These computational findings guide mutation
experiments and enable the identification of mutants with an improved
disproportionation/hydrolysis ratio. The present mechanism is in line
with experimental data, highlighting the critical role of conformational
dynamics in regulating the catalytic reactivity of GHs.