Oligosaccharide Binding in Family 8 Glycosidases: Crystal Structures of Active-Site Mutants of the β-1,4-Xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in Complex with Substrate and Product,

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Abstract
The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-β-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 Å resolution) and product xylotriose (at 1.9 Å 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 2SO conformation.