title: 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.
abstract: 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 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme 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 (2)S(O) conformation.
authors.affiliation: Laboratorium voor Eiwitbiochemie en Eiwitengineering, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium.
authors.forename: D
authors.initials: D
authors.lastname: De Vos
authors.forename: T
authors.initials: T
authors.lastname: Collins
authors.forename: W
authors.initials: W
authors.lastname: Nerinckx
authors.forename: S N
authors.initials: SN
authors.lastname: Savvides
authors.forename: M
authors.initials: M
authors.lastname: Claeyssens
authors.forename: C
authors.initials: C
authors.lastname: Gerday
authors.forename: G
authors.initials: G
authors.lastname: Feller
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002236:Carbohydrate Conformation; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D031161:Pseudoalteromonas / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases