title: Purification, crystallization and preliminary crystallographic analysis of Gan1D, a GH1 6-phospho-β-galactosidase from Geobacillus stearothermophilus T1.
abstract: Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure-function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.
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: Arie
authors.initials: A
authors.lastname: Zehavi
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: Technion Center for Structural Biology, The Lorry I. Lokey Interdisciplinary Center for Life Science 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, and Unit for Virus-Host Cell Interactions, European Synchrotron Radiation Facility, Université Grenoble Alpes-EMBL-CNRS, 6 Rue Jules Horowitz, 38042 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: D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D017931:DNA Primers; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D006026:Glycoside Hydrolases; C022361:6-phospho-beta-galactosidase
keywords: GH1; Geobacillus stearothermophilus; P1; galactan utilization; galactosidases; glycoside hydrolases; high resolution