Preliminary crystallographic analysis of Xyn52B2, a GH52 β-d-xylosidase from Geobacillus stearothermophilus T6

Roie Dann, Shifra Lansky, Noa Lavid, Arie Zehavi, Valery Belakhov, Timor Baasov, Hay Dvir, Babu Manjasetty, Hassan Belrhali, Yuval Shoham, Gil Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P212121 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70Å resolution), the E335G catalytic mutant (2.95Å resolution), a potential mercury derivative (2.15Å resolution) and a selenomethionine derivative (3.90Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Original languageAmerican English
Pages (from-to)1675-1682
Number of pages8
JournalActa Crystallographica Section F:Structural Biology Communications
Volume70
DOIs
StatePublished - 1 Dec 2014

Bibliographical note

Publisher Copyright:
© 2014 International Unoin of Crystallography.

Keywords

  • GH52
  • Geobacillus stearothermophilus
  • SAD data collection
  • enzymatic glycosynthesis
  • glycoside hydrolase
  • glycosynthase
  • selenomethionine
  • xylan utilization
  • xylobiose
  • xylose
  • xylosidase

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