Project description:Xylosidases hydrolyze xylopolymers at the nonreducing end to free xylose units. The ?-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. A BLASTP search with the XylC protein sequence showed that no similar structure had previously been solved. XylC was classified as a member of the new glycoside hydrolase family GH120 according to the CAZy website (http://www.cazy.org/). Crystals belonging to the monoclinic space group P2(1), with unit-cell parameters a = 88.36, b = 202.20, c = 99.87?Å, ? = 99.04°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.2?Å resolution. Structure determination using MIR and MAD methods is in progress.
Project description:The 1,914-bp open reading frame of xylC from Thermoanaerobacterium saccharolyticum JW/SL-YS485 encodes a calculated 73-kDa ?-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third ?-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression of xylC(m) is due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylC(rec)). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylC(rec) exhibited maximum activity at 65°C and pH(65°C) 6.0, a 1-h half-life at 67°C, a K(m) for p-nitrophenyl-?-D-xyloside of 28 mM, and a V(max) of 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.
Project description:Xylan-1,4-?-xylosidase (?-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a ?-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel ?-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a ?-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.
Project description:The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.