Protein crystallization and preliminary determination of three-dimensional structure of rice B-glucosidase (original) (raw)

Rice Os4BGlu12, a glycosyl hydrolase family 1 (GH1) β-glucosidase hydrolyzes β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the β-(1,3)linked disaccharide laminaribiose. Os4BGlu12 was expressed as an N-terminal thioredoxin/His 6 fusion protein in OrigamiB(DE3) Escherichia coli. The fusion protein was purified by immobilized metal-affinity chromatography (IMAC) with cobalt resin. After the thioredoxin/His 6 tag was excised from the protein with enterokinase, the fusion tag was removed by adsorption to the IMAC resin, which yielded a 55 kDa Os4BGlu12 with >95% purity. The free Os4BGlu12 enzyme and a complex with 2,4-dinitrophenyl 2-fluoro-2-deoxy-β-D-glucopyranoside (G2F) inhibitor were crystallized at 15°C. Microbatch crystallization screening generated the native crystals in 25% (w/v) polyethylene glycol (PEG) 4000, 0.1 M Tris-HCl, pH 8.5, 0.20 M NaCl. The conditions were further optimized by the hanging-drop vapordiffusion method with microseeding. Native crystals and crystals of Os4BGlu12 complexed with G2F were obtained in 19% (w/v) PEG 3350, 0.1 M Tris-HCl, pH 8.5, 0.16 M NaCl and 19% (w/v) PEG 2000, 0.1 M Tris-HCl, pH 8.5, 0.16 M NaCl, respectively. Crystals of free Os4BGlu12 and Os4BGlu12-G2F complex were diffracted to 2.50 and 2.45 Å resolution, respectively, and their unit cell symmetry VII determined to be in the tetragonal P4 3 2 1 2 space group. The structure of native Os4BGlu12 was solved by molecular replacement with the 1CBG structure as a search model and had two molecules per asymmetric unit with a solvent content of 49.98% and a Mathews Coefficient (V M ) of 2.46 Å 3 Da -1 . The native Os4BGlu12 structure further served as a template for rigid body refinement to solve the Os4BGlu12 with G2F data set, which had a V M of 2.68 Å 3 Da -1 and 54.17% solvent content. The structures were similar to previous known GH1 enzymes, but the significant differences were seen at the main-chain trace of loop surrounding the active site. The active site is located at the bottom of an approximately 20 Å deep slot-like pocket surrounded by a large surface loop. In the innermost part of the active site in the crystal structure of G2F complex, the surrounding conserved amino acid residues seen in other GH1 enzymes formed hydrogen bonds with the glucosyl unit.