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Peer-reviewedCC BY 4.0The Febs Journal

Cryo‐EM structure of GH43 β‐Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity

  • Lorenzo Briganti1
  • Andre Schutzer Godoy1,2
  • Caio Cesar Mello Capetti1
  • Rafaela Sachetto Fernandes1
  • Vanessa O. A. Pellegrini1
  • Ornella M. Ontañon3
  • Eleonora Campos3
  • Rodrigo V. Portugal4
  • Igor Polikarpov1
4 affiliations
  1. 1São Carlos Institute of Physics University of São Paulo Brazil
  2. 2Echo Life Sciences São Carlos Brazil
  3. 3Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CONICET‐INTA Prov. Buenos Aires Argentina
  4. 4Brazilian Nanotechnology National Laboratory CNPEM Campinas São Paulo Brazil
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Abstract

Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β‐1,4‐linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β‐xylanases and β‐xylosidases. In this study, we determined the cryo‐electron microscopy (cryo‐EM) structure of Enterobacter cloacae β‐xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X‐ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β‐xylosidase from the Enterobacter genus using both X‐ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.

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Cite

BibTeXRIS
BibTeX
@article{briganti2026cryoem,
  title = {{Cryo‐EM structure of GH43 β‐Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity}},
  author = {Lorenzo Briganti and Andre Schutzer Godoy and Caio Cesar Mello Capetti and Rafaela Sachetto Fernandes and Vanessa O. A. Pellegrini and Ornella M. Ontañon and Eleonora Campos and Rodrigo V. Portugal and Igor Polikarpov},
  journal = {The Febs Journal},
  year = {2026},
  month = may,
  doi = {10.1111/febs.70590},
  url = {https://doi.org/10.1111/febs.70590},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Cryo‐EM structure of GH43 β‐Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
AU  - Lorenzo Briganti
AU  - Andre Schutzer Godoy
AU  - Caio Cesar Mello Capetti
AU  - Rafaela Sachetto Fernandes
AU  - Vanessa O. A. Pellegrini
AU  - Ornella M. Ontañon
AU  - Eleonora Campos
AU  - Rodrigo V. Portugal
AU  - Igor Polikarpov
PY  - 2026
DA  - 2026/05/18
JO  - The Febs Journal
DO  - 10.1111/febs.70590
UR  - https://doi.org/10.1111/febs.70590
AB  - Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β‐1,4‐linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β‐xylanases and β‐xylosidases. In this study, we determined the cryo‐electron microscopy (cryo‐EM) structure of Enterobacter cloacae β‐xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X‐ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β‐xylosidase from the Enterobacter genus using both X‐ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.
SN  - 1742-464X
SN  - 1742-4658
ER  -