Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular β-galactosidase fromGeobacillus stearothermophilus

Abstract

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a multi-enzyme system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo-acting extracellular enzymes that break down the highmolecular-weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 -galactosidase capable of hydrolyzing short -1,4-galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure-function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P2 1 2 1 2 1 , with average crystallographic unit-cell parameters of a = 71.84, b = 181.35, c = 196.57 Å . Full diffraction data sets to 2.45 and 2.50 Å resolution have been collected for both the wild-type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash-cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three-dimensional crystal structure determination of GanB.

Structure–function relationships in Gan42B, an intracellular GH42 β-galactosidase fromGeobacillus stearothermophilus

“…The crystallization conditions and final sizes and shapes of the resulting crystals were closely similar for Gan42B-WT and Gan42B-E323A. For unknown reasons both crystals were very hard to reproduce, and usually only a small percentage of the hanging drops resulted in crystallographically useful crystals (Solomon et al, 2013).…”

“…The ganB gene with a tag of six histidine residues fused at its N-terminus was cloned into the expression vector pET-9d as described previously (Tabachnikov & Shoham, 2013;Solomon et al, 2013). Briefly, site-directed mutagenesis of ganB was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California, USA).…”

“…As described previously in greater detail (Solomon et al, 2013), suitable crystals of the purified Gan42B-WT and Gan42B-E323A proteins (including their original His tags) were obtained by a series of hanging-drop vapour-diffusion experiments, optimizing parameters such as the pH, ionic strength, protein concentration and temperature (Almog et al, 1993(Almog et al, , 1994Gilboa et al, 1998;Golan, Zharkov et al, 2004;Reiland et al, 2004), as well as precipitating additives (Lansky, Salama, Solomon et al, 2013;Lansky, Zehavi et al, 2014). For both proteins, the best diffracting crystals were obtained at 21 C in 4 ml drops consisting of a mixture of 2 ml protein solution (18-22 mg ml À1 in 100 mM NaCl, 0.02% sodium azide, 50 mM Tris buffer pH 7.0) and 2 ml crystallization reservoir solution (16-18% PEG 3350, 250 mM NaCl, 100 mM HEPES buffer pH 7.0).…”

“…In all cases it was necessary to separate the target crystal from the multi-crystal cluster obtained in order to submit it to full crystallographic analysis. This was performed by mechanical cutting of the cluster into separate plates, as close as possible to the cluster-connecting part of the plate, and with special efforts to apply minimal deformation force to the cluster and the separated crystals (Solomon et al, 2013). The crystal flash-cooling procedure included a short 20-30 s presoaking of the selected crystal in a cryoprotecting solution, consisting of 87% of the original crystallization reservoir solution and 13%(v/v) glycerol, prior to its quick immersion in liquid nitrogen.…”

Choosing the Method of Crystallization to Obtain Optimal Results

“…Further examples where placing an oil barrier over the reservoir dramatically reduced the nucleation, were the rat liver ribosomal P2 protein thereby resulting in higher diffracting crystals, with a resolution limit of 2.4 Å ( Figure 2) [18] and GH42 intracellular beta-galactosidase diffracting to 2.45Å [19]. At the moment, such trials can only be done manually, but an oil barrier technique, which is miniaturized and automated, will soon be published.…”