Donor-induced conformational gating and substrate-assisted catalysis in α-1,3-galactosyltransferase
Linares-Pastén, Javier A. LU
and Planas, Antoni
(2026)
In Protein Science
35(9).
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/373a5bdc-3a86-4981-9089-e0c40d687866
- author
- Linares-Pastén, Javier A.
LU
and Planas, Antoni
- organization
- publishing date
- 2026-08-15
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Protein Science
- volume
- 35
- issue
- 9
- article number
- e70770
- pages
- 13 pages
- publisher
- The Protein Society
- external identifiers
-
- pmid:42603117
- ISSN
- 1469-896X
- DOI
- 10.1002/pro.70770
- language
- English
- LU publication?
- yes
- id
- 373a5bdc-3a86-4981-9089-e0c40d687866
- date added to LUP
- 2026-08-17 22:49:27
- date last changed
- 2026-08-21 14:19:39
@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}},
}