@article{373a5bdc-3a86-4981-9089-e0c40d687866,
  abstract     = {{Retaining glycosyltransferases catalyze the formation of stereochemically conserved glycosidic bonds through mechanisms that remain debated. Using bovine α1,3-galactosyltransferase (α3GalT) as a model, we combine mutagenesis, equilibrium unfolding, kinetics, and molecular dynamics simulations to understand how donor-induced loop ordering promotes catalysis. Alanine-scanning mutagenesis of the C-terminal loop (Thr358-Val368) identified Lys359, Tyr361, and Arg365 as critical for donor binding, catalysis, and ligand-dependent stabilization. In addition, D225A and E317A were inactive and showed minimal ligand-induced stabilization, consistent with impaired metal binding and substrate stabilization, respectively. Donor binding induces an ordered conformation in the C-terminus, reducing its local flexibility by 30% and pre-organizing the active site for catalysis. MD-derived energy profiles differed markedly for the donor (UDP-Gal) and acceptor (lactose) in the ternary complex. In this context, experimental apparent Kₘ values indicate higher donor affinity than acceptor affinity. Our results show that donor binding stabilizes the C-terminal loop, assembling a competent complex for catalysis. These findings support a general coupling between conformational gating, donor stabilization, and the catalytic mechanism in retaining GT-A-fold enzymes.}},
  author       = {{Linares-Pastén, Javier A. and Planas, Antoni}},
  issn         = {{1469-896X}},
  language     = {{eng}},
  month        = {{08}},
  number       = {{9}},
  publisher    = {{The Protein Society}},
  series       = {{Protein Science}},
  title        = {{Donor-induced conformational gating and substrate-assisted catalysis in <i>α</i>-1,3-galactosyltransferase}},
  url          = {{http://dx.doi.org/10.1002/pro.70770}},
  doi          = {{10.1002/pro.70770}},
  volume       = {{35}},
  year         = {{2026}},
}