title: Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase from Geobacillus stearothermophilus T6.
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 = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Roie
authors.initials: R
authors.lastname: Dann
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Shifra
authors.initials: S
authors.lastname: Lansky
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Noa
authors.initials: N
authors.lastname: Lavid
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Arie
authors.initials: A
authors.lastname: Zehavi
authors.affiliation: Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Valery
authors.initials: V
authors.lastname: Belakhov
authors.affiliation: Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Timor
authors.initials: T
authors.lastname: Baasov
authors.affiliation: Technion Center for Structural Biology, Lorry I. Lokey Center for Life Sciences and Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Hay
authors.initials: H
authors.lastname: Dvir
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38000 Grenoble, France.
authors.forename: Babu
authors.initials: B
authors.lastname: Manjasetty
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38000 Grenoble, France.
authors.forename: Hassan
authors.initials: H
authors.lastname: Belrhali
authors.affiliation: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Gil
authors.initials: G
authors.lastname: Shoham
mesh_terms: D003461:Crystallography; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D011487:Protein Conformation; D014995:Xylosidases / Q000737:chemistry*
chemical_list: D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: GH52; Geobacillus stearothermophilus; SAD data collection; enzymatic glycosynthesis; glycoside hydrolase; glycosynthase; selenomethionine; xylan utilization; xylobiose; xylose; xylosidase