The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit
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The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit. / Paës, Gabriel; Skov, Lars K; O'Donohue, Michael J; Rémond, Caroline; Kastrup, Jette Sandholm; Gajhede, Michael; Mirza, Osman.
In: Biochemistry, Vol. 47, No. 28, 2008, p. 7441-7451.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit
AU - Paës, Gabriel
AU - Skov, Lars K
AU - O'Donohue, Michael J
AU - Rémond, Caroline
AU - Kastrup, Jette Sandholm
AU - Gajhede, Michael
AU - Mirza, Osman
N1 - Keywords: Arabinose; Bacillaceae; Bacterial Proteins; Carbohydrate Conformation; Crystallography, X-Ray; Glycoside Hydrolases; Models, Molecular; Mutagenesis, Site-Directed; Oligosaccharides; Recombinant Proteins; Thermodynamics; Xylans
PY - 2008
Y1 - 2008
N2 - The crystal structure of the family GH-51 alpha- l-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural substrate xylan. The overall structure shows the two characteristic GH-51 domains: a catalytic domain that is folded into a (beta/alpha) 8-barrel and a C-terminal domain that displays jelly roll architecture. The pentasaccharide is bound in a groove on the surface of the enzyme, with the mono arabinosyl branch entering a tight pocket harboring the catalytic dyad. Detailed analyses of both structures and comparisons with the two previously determined structures from Geobacillus stearothermophilus and Clostridium thermocellum reveal important details unique to the Thermobacillus xylanilyticus enzyme. In the absence of substrate, the enzyme adopts an open conformation. In the substrate-bound form, the long loop connecting beta-strand 2 to alpha-helix 2 closes the active site and interacts with the substrate through residues His98 and Trp99. The results of kinetic and fluorescence titration studies using mutants underline the importance of this loop, and support the notion of an interaction between Trp99 and the bound substrate. We suggest that the changes in loop conformation are an integral part of the T. xylanilyticus alpha- l-arabinofuranosidase reaction mechanism, and ensure efficient binding and release of substrate.
AB - The crystal structure of the family GH-51 alpha- l-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural substrate xylan. The overall structure shows the two characteristic GH-51 domains: a catalytic domain that is folded into a (beta/alpha) 8-barrel and a C-terminal domain that displays jelly roll architecture. The pentasaccharide is bound in a groove on the surface of the enzyme, with the mono arabinosyl branch entering a tight pocket harboring the catalytic dyad. Detailed analyses of both structures and comparisons with the two previously determined structures from Geobacillus stearothermophilus and Clostridium thermocellum reveal important details unique to the Thermobacillus xylanilyticus enzyme. In the absence of substrate, the enzyme adopts an open conformation. In the substrate-bound form, the long loop connecting beta-strand 2 to alpha-helix 2 closes the active site and interacts with the substrate through residues His98 and Trp99. The results of kinetic and fluorescence titration studies using mutants underline the importance of this loop, and support the notion of an interaction between Trp99 and the bound substrate. We suggest that the changes in loop conformation are an integral part of the T. xylanilyticus alpha- l-arabinofuranosidase reaction mechanism, and ensure efficient binding and release of substrate.
KW - Former Faculty of Pharmaceutical Sciences
U2 - 10.1021/bi800424e
DO - 10.1021/bi800424e
M3 - Journal article
C2 - 18563919
VL - 47
SP - 7441
EP - 7451
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 28
ER -
ID: 9937210