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Research article - Peer-reviewed, 2003

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D

von Ossowski I, Stahlberg J, Koivula A, Piens K, Becker D, Boer H, Harle R, Harris M, Divne C, Mahdi S, Zhao YX, Driguez H, Claeyssens M, Sinnott ML, Teeri TT


The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop. (C) 2003 Elsevier Ltd. All rights reserved


cellulose; crystal structure; glycoside hydrolase; processivity; product inhibition

Published in

Journal of Molecular Biology
2003, volume: 333, number: 4, pages: 817-829

Authors' information

Swedish University of Agricultural Sciences, Department of Molecular Biology
Becker, Dieter
Boer, Harry
Claeyssens, Marc
Divne, Christina
Driguez, Hugues
Harle, Raija
Harris, Mark
Koivula, Anu
Sinnott, Michael L
von Ossowski, Ingemar
Zhao, Yongxin
Piens, Kathleen
Teeri, Tuula T
Mahdi, Sabah
Swedish University of Agricultural Sciences, Department of Molecular Biology

UKÄ Subject classification

Renewable Bioenergy Research

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