Crystallization and preliminary X-ray crystallographic analysis of β-xylosidase fromThermoanaerobacterium saccharolyticum, a thermophilic anaerobe

Abstract

-Xylosidases are involved in the breakdown of xylans into xylose and belong to either family 39 or 43 of the glycosyl hydrolases. At present, no structural information is available for any member of these families. -Xylosidase from the thermophilic anaerobe Thermoanaerobacterium saccharolyticum, a member of glycosyl hydrolase family 39, has been crystallized at 296 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.4 A Ê resolution with synchrotron X-rays and belongs to space group 4 1 2 1 2 (or P4 3 2 1 2), with unit-cell parameters a = b = 92.75, c = 241.37 A Ê . The asymmetric unit contains two monomers of the recombinant enzyme, giving a corresponding V M of 2.21 A Ê 3 Da À1 and a solvent content of 44.3%.

Crystal Structure of β-d-Xylosidase from Thermoanaerobacterium saccharolyticum, a Family 39 Glycoside Hydrolase

“…The SeMet-substituted xylosidase was crystallized under the same conditions as the native protein. 13 The SeMet-substituted crystal belongs to the same space group (i.e. P4 3 2 1 2) as the native protein crystal, with unit cell parameters of a ¼ b ¼ 92.72 Å , c ¼ 241.30 Å , and a ¼ b ¼ g ¼ 908.…”

“…2,31 Purification and crystallization of the native protein into form A has been described. 13 The SeMet-substituted xylosidase was expressed using the M9 cell culture medium, in which methionine is substituted with SeMet; 10 mM dithiothreitol was added during purification. The SeMet-substituted xylosidase was crystallized under the same conditions as the native protein.…”

“…9 Results and Discussion Structure determination T. saccharolyticum b-xylosidase crystallized into two different crystal forms under similar crystallization conditions: form A (space group P4 3 2 1 2, with two monomers per asymmetric unit) and form B (P2 1 2 1 2 1 , with four monomers per asymmetric unit). It crystallized into form A in the presence of D-xylose, 13 whereas it crystallized into form B when it was incubated with the inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-b-D-xyloside (2F-DNPX). 14 The structure of T. saccharolyticum b-xylosidase was determined using multiwavelength anomalous diffraction (MAD) data collected to 2.4 Å from a selenomethionine (SeMet)-labeled crystal of form A (Table 1).…”

“…Crystallization of a fungal b-xylosidase from Trichoderma reesei, a member of GH family 3 and T. saccharolyticum b-xylosidase has been reported. 12,13 To obtain structural information necessary for understanding the catalytic mechanism, we have determined the crystal structure of T. saccharolyticum b-xylosidase in two forms: a D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-a-D-xylosyl (2F-xylosyl)-xylosidase intermediate. The structure of the covalent reaction intermediate provides direct structural evidence supporting the proposed catalytic mechanism and provides insight into other residues involved in catalysis.…”