Article (Scientific journals)
Study of the active site residues of a glycoside hydrolase family 8 xylanase
Collins, T.; De Vos, D.; Hoyoux, A. et al.
2005In Journal of Molecular Biology, 354 (2), p. 425-435
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Keywords :
catalytic site residues; glycoside hydrolase family 8; xylanase
Abstract :
[en] Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family. (c) 2005 Elsevier Ltd. All rights reserved.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Collins, T.
De Vos, D.
Hoyoux, A.
Savvides, S. N.
Gerday, Charles ;  Université de Liège - ULiège > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences)
Van Beeumen, J.
Feller, Georges ;  Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Language :
English
Title :
Study of the active site residues of a glycoside hydrolase family 8 xylanase
Publication date :
25 November 2005
Journal title :
Journal of Molecular Biology
ISSN :
0022-2836
eISSN :
1089-8638
Publisher :
Academic Press Ltd Elsevier Science Ltd, London, United Kingdom
Volume :
354
Issue :
2
Pages :
425-435
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 26 January 2010

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