Enzyme-substrate complex structures of a GH39 β-xylosidase from Geobacillus stearothermophilus

Mirjam Czjzek*, Alon Ben David, Tsafrir Bravman, Gil Shoham, Bernard Henrissat, Yuval Shoham

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

β-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. β-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 β-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (β/α)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus β-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-β-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.

Original languageAmerican English
Pages (from-to)838-846
Number of pages9
JournalJournal of Molecular Biology
Volume353
Issue number4
DOIs
StatePublished - 4 Nov 2005

Bibliographical note

Funding Information:
This study was supported by grants from the French-Israeli Association for Scientific and Technological Research (AFIRST) (to Y.S. and B.H), G.I.F., the German-Israeli-Foundation for Scientific Research and Development (to Y.S. and G.S.), and from the Israel Science Foundation (to G.S. and Y.S.). Additional support was provided by the Otto Meyerhof Center for Biotechnology, Technion, established by the Minerva Foundation (Munich, Germany). We are indebted to the ESRF for beam-time allocation for this project and to the staff on beamlines ID14-EH2 and ID14-EH4 for technical assistance during data collections.

Keywords

  • Covalent reaction intermediate
  • Crystal structure
  • Enzyme-substrate complex
  • GH39
  • β-xylosidase

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