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Purification, crystallization and preliminary crystallographic analysis of Gan1D, a GH1 6‐phospho‐β‐galactosidase from Geobacillus stearothermophilus T1
Author(s) -
Lansky Shifra,
Zehavi Arie,
Dann Roie,
Dvir Hay,
Belrhali Hassan,
Shoham Yuval,
Shoham Gil
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x13034778
Subject(s) - geobacillus stearothermophilus , thermophile , glycoside hydrolase , cell wall , biochemistry , polysaccharide , triclinic crystal system , enzyme , chemistry , crystallography , biology , crystal structure
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 P 1, 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.