The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala224) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Å (= 0.1nm) closer to the acid/base Glu217 residue, with a 3.1 Å contact between N∊2 and O∊1. The pH variation of kcat/Km for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK1 shifting from 2.22±0.03 in the wild-type to 3.19±0.03 in the mutant, and pK2 shifting from 5.99±0.02 to 6.78±0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative kcat for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower kcat/Km values for both lactosides and cellobiosides, and a marginally lower stability. However, kcat values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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Research Article|
May 08 2001
Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant
Dieter BECKER;
Dieter BECKER
∗Department of Paper Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Sackville Street, Manchester M60 lQD, U.K.
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Christophe BRAET;
Christophe BRAET
†Department of Biochemistry, Physiology and Microbiology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
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Harry BRUMER, III;
Harry BRUMER, III
‡Department of Biotechnology, Royal Institute of Technology, S-10044 Stockholm, Sweden
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Marc CLAEYSSENS;
Marc CLAEYSSENS
†Department of Biochemistry, Physiology and Microbiology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
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Christina DIVNE;
Christina DIVNE
∥Department of Cell and Molecular Biology, Uppsala University, BMC, P.O. Box 596, SE-75124, Uppsala, Sweden
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B. Richard FAGERSTRÖM;
B. Richard FAGERSTRÖM
§Röhm Enzyme Finland OY, PL 26, Tykkimäentie 15, FIN-05200 Rajamäki, Finland
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Mark HARRIS;
Mark HARRIS
∥Department of Cell and Molecular Biology, Uppsala University, BMC, P.O. Box 596, SE-75124, Uppsala, Sweden
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T. Alwyn JONES;
T. Alwyn JONES
∥Department of Cell and Molecular Biology, Uppsala University, BMC, P.O. Box 596, SE-75124, Uppsala, Sweden
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Gerard J. KLEYWEGT;
Gerard J. KLEYWEGT
∥Department of Cell and Molecular Biology, Uppsala University, BMC, P.O. Box 596, SE-75124, Uppsala, Sweden
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Anu KOIVULA;
Anu KOIVULA
¶VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
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Sabah MAHDI;
Sabah MAHDI
∗∗Department of Molecular Biology, Swedish University of Agricultural Sciences, BMC, P.O. Box 590, SE-75124, Uppsala, Sweden
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Kathleen PIENS;
Kathleen PIENS
†Department of Biochemistry, Physiology and Microbiology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
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Michael L. SINNOTT;
Michael L. SINNOTT
1
∗Department of Paper Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Sackville Street, Manchester M60 lQD, U.K.
1To whom correspondence should be addressed (e-mail: Michael.Sinnott@umist.ac.uk)
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Jerry STÅHLBERG;
Jerry STÅHLBERG
∗∗Department of Molecular Biology, Swedish University of Agricultural Sciences, BMC, P.O. Box 590, SE-75124, Uppsala, Sweden
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Tuula T. TEERI;
Tuula T. TEERI
‡Department of Biotechnology, Royal Institute of Technology, S-10044 Stockholm, Sweden
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Melanie UNDERWOOD;
Melanie UNDERWOOD
∗Department of Paper Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Sackville Street, Manchester M60 lQD, U.K.
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Gerd WOHLFAHRT
Gerd WOHLFAHRT
¶VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
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Publisher: Portland Press Ltd
Received:
July 13 2000
Revision Received:
January 26 2001
Accepted:
March 02 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 356 (1): 19–30.
Article history
Received:
July 13 2000
Revision Received:
January 26 2001
Accepted:
March 02 2001
Citation
Dieter BECKER, Christophe BRAET, Harry BRUMER, Marc CLAEYSSENS, Christina DIVNE, B. Richard FAGERSTRÖM, Mark HARRIS, T. Alwyn JONES, Gerard J. KLEYWEGT, Anu KOIVULA, Sabah MAHDI, Kathleen PIENS, Michael L. SINNOTT, Jerry STÅHLBERG, Tuula T. TEERI, Melanie UNDERWOOD, Gerd WOHLFAHRT; Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant. Biochem J 15 May 2001; 356 (1): 19–30. doi: https://doi.org/10.1042/bj3560019
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