The Structure of an Inverting GH43 β-Xylosidase from Geobacillus stearothermophilus with its Substrate Reveals the Role of the Three Catalytic Residues

Christian Brüx, Alon Ben-David, Dalia Shallom-Shezifi, Maya Leon, Karsten Niefind, Gil Shoham, Yuval Shoham*, Dietmar Schomburg

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

124 Scopus citations


β-d-Xylosidases are glycoside hydrolases that catalyze the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicellulose. Here we describe the enzyme-substrate crystal structure of an inverting family 43 β-xylosidase, from Geobacillus stearothermophilus T-6 (XynB3). Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed β-propeller catalytic domain, and a β-sandwich domain. The active site possesses a pocket topology, which is mainly constructed from the β-propeller domain residues, and is closed on one side by a loop that originates from the β-sandwich domain. This loop restricts the length of xylose units that can enter the active site, consistent with the exo mode of action of the enzyme. Structures of the enzyme-substrate (xylobiose) complex provide insights into the role of the three catalytic residues. The xylose moiety at the -1 subsite is held by a large number of hydrogen bonds, whereas only one hydroxyl of the xylose unit at the +1 subsite can create hydrogen bonds with the enzyme. The general base, Asp15, is located on the α-side of the -1 xylose sugar ring, 5.2 Å from the anomeric carbon. This location enables it to activate a water molecule for a single-displacement attack on the anomeric carbon, resulting in inversion of the anomeric configuration. Glu187, the general acid, is 2.4 Å from the glycosidic oxygen atom and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Å from the general acid; modulating its pKa and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is an invariant among all five-bladed β-propeller glycoside hydrolases.

Original languageAmerican English
Pages (from-to)97-109
Number of pages13
JournalJournal of Molecular Biology
Issue number1
StatePublished - 26 May 2006

Bibliographical note

Funding Information:
This study was supported by grants from the G.I.F., the German-Israeli-Foundation for Scientific Research and Development (to Y.S., D.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 also thank the staff of the EMBL Hamburg Outstation for excellent technical support.


  • crystal structure
  • enzyme-substrate complex
  • glycoside hydrolase family 43
  • substrate specificity
  • β-xylosidase


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