Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their amino acid sequence similarities. Endoglucanase I (Cel7B), from the soft-rot fungus Humicola insolens, is a family 7 enzyme. The structure of the native form of Cel7B from H. insolens at 2.2 Å resolution has been solved by molecular replacement using the known Trichoderma reesei cellobiohydrolase I [Divne, Ståhlberg, Reinikainen, Ruohonen, Pettersson, Knowles, Teeri and Jones (1994) Science265, 524–528] structure as the search model. Cel7B catalyses hydrolysis of the β-1,4 glycosidic linkages in cellulose with net retention of anomeric configuration. The catalytic nucleophile at the active site of Cel7B has been identified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzyme intermediate and identification of the labelled peptide in peptic digests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, confirming the critical role of these groups for catalysis.
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Research Article|
October 15 1998
Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 Å resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate
Lloyd F. MACKENZIE;
Lloyd F. MACKENZIE
*Department of Chemistry, University of British Columbia, Vancouver, V6T 1Z1, Canada
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Gerlind SULZENBACHER;
Gerlind SULZENBACHER
†Department of Chemistry, University of York, Heslington, York Y01 5DD, U.K.
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Christina DIVNE;
Christina DIVNE
‡Department of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden
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T. Alwyn JONES;
T. Alwyn JONES
‡Department of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden
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Helle F. WÖLDIKE;
Helle F. WÖLDIKE
§Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark
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Martin SCHÜLEIN;
Martin SCHÜLEIN
§Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark
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Stephen G. WITHERS;
Stephen G. WITHERS
*Department of Chemistry, University of British Columbia, Vancouver, V6T 1Z1, Canada
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Gideon J. DAVIES
Gideon J. DAVIES
1
†Department of Chemistry, University of York, Heslington, York Y01 5DD, U.K.
1To whom correspondence should be addressed (davies@yorvic.york.ac.uk).
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Publisher: Portland Press Ltd
Received:
May 22 1998
Revision Received:
July 16 1998
Accepted:
August 07 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 335 (2): 409–416.
Article history
Received:
May 22 1998
Revision Received:
July 16 1998
Accepted:
August 07 1998
Citation
Lloyd F. MACKENZIE, Gerlind SULZENBACHER, Christina DIVNE, T. Alwyn JONES, Helle F. WÖLDIKE, Martin SCHÜLEIN, Stephen G. WITHERS, Gideon J. DAVIES; Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 Å resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem J 15 October 1998; 335 (2): 409–416. doi: https://doi.org/10.1042/bj3350409
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