Oligosaccharide binding in family 8 glycosidases : crystal structures of active-site mutants of the <script type="math/tex">\beta</script>-1,4-xylanase pXyl from **Pseudoaltermonas haloplanktis** TAH3a in complex with substrate and product

De Vos, D.; Collins, T.; Nerinckx, W.; Savvides, S.N.; Claeyssens, M.; Gerday, C.; Feller, G.; Van Beeumen, J.

doi:10.1021/BI052193E

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

Author

De Vos, D.

Collins, T.

Nerinckx, W.

Savvides, S.N.

Claeyssens, M.

Gerday, C.

Feller, G.

Van Beeumen, J.

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.

Language

English

Source (journal)

Biochemistry / American Chemical Society. - Washington, D.C., 1962, currens

Publication

Washington, D.C. : 2006

ISSN

0006-2960 [print]

1520-4995 [online]

DOI

10.1021/BI052193E

Volume/pages