Oligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product

• Published in: Biochemistry
• Main Authors: De Vos, D., Collins, T., Nerinckx, W., Savvides, S. N., Claeyssens, M., Gerday, Charles, Feller, Georges, Van Beeumen, J.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Amer Chemical Soc 2006
• Subjects: Life sciences; Biochemistry; biophysics & molecular biology; Sciences du vivant; Biochimie; biophysique & biologie moléculaire; Antarc*; Antarctic
• Online Access: https://orbi.uliege.be/handle/2268/15381; https://doi.org/10.1021/bi052193e

peer reviewed The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 angstrom resolution) and product xylotriose (at 1.9 angstrom resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apoenzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position Occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking Studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum S-2(O) conformation.