Complementary chemical probes and substrates reveal split subsite specificity in a fungal GH12 glucanase
8 affiliations
- 1Department of Chemistry
- 2University of York
- 3Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, Leiden, Netherlands, 2333 CC
- 4INRAE
- 5Aix-Marseille Université, BBF, Biodiversité et Biotechnologie Fongiques
- 6CERMAV
- 7TBI
- 8Université de Toulouse, CNRS, INRAE, INSA
Abstract
Xyloglucans are structurally heterogeneous polysaccharides in which xylosylation patterns vary along the β-(1,4)-glucan backbone. Because endo-glycanases engage substrates across both sides of the scissile bond, we hypothesised that GH12 substrate selectivity may vary across the catalytic cleft. We combined activity-based profiling of basidiomycete secretomes with defined fluorogenic substrates, oligosaccharide product mapping and structural analyses. TlGH12A, a previously characterised endo-β-glucanase, reacted with a xyloglucan-configured cyclophellitol probe despite showing no detectable activity on polymeric xyloglucan. Complementary analyses revealed that substrate recognition differs across its catalytic cleft. TlGH12A hydrolysed XXXG-4MU approximately 60-fold more efficiently than GGGG-4MU, demonstrating a strong negative-subsite preference for xylosylated glucan. Crystal structures showed xylosylated glucan occupying subsites −4 to −1, whereas unbranched glucan occupied the positive subsites. Comparison with ligand-bound GH12 xyloglucanases identified steric constraints that may restrict positive-subsite accommodation of Xyl substituents. Sequence-network, phylogenetic and structural-modelling analyses suggest that this architecture recurs among Basidiomycota GH12 endoglucanases. Complementary probes, hydrolysable substrates and structural analyses resolved the basis of this previously unrecognised distribution of specificity in TlGH12A. These findings reveal how substrate recognition can vary across a GH12 catalytic cleft, with implications for understanding fungal plant cell wall deconstruction and for identifying and engineering glucan-active enzymes with distinct substrate specificities.
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BibTeX
@misc{correa2026complementary,
title = {{Complementary chemical probes and substrates reveal split subsite specificity in a fungal GH12 glucanase}},
author = {Thamy L R Corrêa and Nicholas GS McGregor and Casper de Boer and Sybrin P. Schröder and Quentin Foucart and Lianne I Willems and Jean-Guy Berrin and Régis Fauré and Herman S Overkleeft and Gideon J Davies},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15009200/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009200/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Complementary chemical probes and substrates reveal split subsite specificity in a fungal GH12 glucanase
AU - Thamy L R Corrêa
AU - Nicholas GS McGregor
AU - Casper de Boer
AU - Sybrin P. Schröder
AU - Quentin Foucart
AU - Lianne I Willems
AU - Jean-Guy Berrin
AU - Régis Fauré
AU - Herman S Overkleeft
AU - Gideon J Davies
PY - 2026
DA - 2026/09/21
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009200/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009200/v1
AB - Xyloglucans are structurally heterogeneous polysaccharides in which xylosylation patterns vary along the β-(1,4)-glucan backbone. Because endo-glycanases engage substrates across both sides of the scissile bond, we hypothesised that GH12 substrate selectivity may vary across the catalytic cleft. We combined activity-based profiling of basidiomycete secretomes with defined fluorogenic substrates, oligosaccharide product mapping and structural analyses. TlGH12A, a previously characterised endo-β-glucanase, reacted with a xyloglucan-configured cyclophellitol probe despite showing no detectable activity on polymeric xyloglucan. Complementary analyses revealed that substrate recognition differs across its catalytic cleft. TlGH12A hydrolysed XXXG-4MU approximately 60-fold more efficiently than GGGG-4MU, demonstrating a strong negative-subsite preference for xylosylated glucan. Crystal structures showed xylosylated glucan occupying subsites −4 to −1, whereas unbranched glucan occupied the positive subsites. Comparison with ligand-bound GH12 xyloglucanases identified steric constraints that may restrict positive-subsite accommodation of Xyl substituents. Sequence-network, phylogenetic and structural-modelling analyses suggest that this architecture recurs among Basidiomycota GH12 endoglucanases. Complementary probes, hydrolysable substrates and structural analyses resolved the basis of this previously unrecognised distribution of specificity in TlGH12A. These findings reveal how substrate recognition can vary across a GH12 catalytic cleft, with implications for understanding fungal plant cell wall deconstruction and for identifying and engineering glucan-active enzymes with distinct substrate specificities.
ER -