Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A

A comparison with Phanerochaete chrysosporium Cel7D

Ingemar von Ossowski, Jerry Ståhlberg, Anu Koivula, Kathleen Piens, Dieter Becker, Harry Boer, Raija Harle, Mark Harris, Christina Divne, Sabah Mahdi, Yongxin Zhao, Hugues Driguez, Marc Claeyssens, Michael L. Sinnott, Tuula Teeri (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

117 Citations (Scopus)

Abstract

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 Δ(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 kcat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both kcat and KM 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 Glc2/(Glc3+Glc1) 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.
Original languageEnglish
Pages (from-to)817-829
Number of pages13
JournalJournal of Molecular Biology
Volume333
Issue number4
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Phanerochaete
Trichoderma
Cellulose
Disulfides
Mutation
Enzymes
Tyrosine
Hydrogen
Catalytic Domain
Hydrolysis

Keywords

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

Cite this

von Ossowski, Ingemar ; Ståhlberg, Jerry ; Koivula, Anu ; Piens, Kathleen ; Becker, Dieter ; Boer, Harry ; Harle, Raija ; Harris, Mark ; Divne, Christina ; Mahdi, Sabah ; Zhao, Yongxin ; Driguez, Hugues ; Claeyssens, Marc ; Sinnott, Michael L. ; Teeri, Tuula. / Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A : A comparison with Phanerochaete chrysosporium Cel7D. In: Journal of Molecular Biology. 2003 ; Vol. 333, No. 4. pp. 817-829.
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title = "Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A: A comparison with Phanerochaete chrysosporium Cel7D",
abstract = "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 Δ(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 kcat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both kcat and KM 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 Glc2/(Glc3+Glc1) 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.",
keywords = "cellulose, crystal structure, glycoside hydrolase, processivity, product inhibition",
author = "{von Ossowski}, Ingemar and Jerry St{\aa}hlberg and Anu Koivula and Kathleen Piens and Dieter Becker and Harry Boer and Raija Harle and Mark Harris and Christina Divne and Sabah Mahdi and Yongxin Zhao and Hugues Driguez and Marc Claeyssens and Sinnott, {Michael L.} and Tuula Teeri",
year = "2003",
doi = "10.1016/S0022-2836(03)00881-7",
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von Ossowski, I, Ståhlberg, J, Koivula, A, Piens, K, Becker, D, Boer, H, Harle, R, Harris, M, Divne, C, Mahdi, S, Zhao, Y, Driguez, H, Claeyssens, M, Sinnott, ML & Teeri, T 2003, 'Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A: A comparison with Phanerochaete chrysosporium Cel7D', Journal of Molecular Biology, vol. 333, no. 4, pp. 817-829. https://doi.org/10.1016/S0022-2836(03)00881-7

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A : A comparison with Phanerochaete chrysosporium Cel7D. / von Ossowski, Ingemar; Ståhlberg, Jerry; Koivula, Anu; Piens, Kathleen; Becker, Dieter; Boer, Harry; Harle, Raija; Harris, Mark; Divne, Christina; Mahdi, Sabah; Zhao, Yongxin; Driguez, Hugues; Claeyssens, Marc; Sinnott, Michael L.; Teeri, Tuula (Corresponding Author).

In: Journal of Molecular Biology, Vol. 333, No. 4, 2003, p. 817-829.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A

T2 - A comparison with Phanerochaete chrysosporium Cel7D

AU - von Ossowski, Ingemar

AU - Ståhlberg, Jerry

AU - Koivula, Anu

AU - Piens, Kathleen

AU - Becker, Dieter

AU - Boer, Harry

AU - Harle, Raija

AU - Harris, Mark

AU - Divne, Christina

AU - Mahdi, Sabah

AU - Zhao, Yongxin

AU - Driguez, Hugues

AU - Claeyssens, Marc

AU - Sinnott, Michael L.

AU - Teeri, Tuula

PY - 2003

Y1 - 2003

N2 - 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 Δ(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 kcat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both kcat and KM 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 Glc2/(Glc3+Glc1) 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.

AB - 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 Δ(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 kcat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both kcat and KM 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 Glc2/(Glc3+Glc1) 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.

KW - cellulose

KW - crystal structure

KW - glycoside hydrolase

KW - processivity

KW - product inhibition

U2 - 10.1016/S0022-2836(03)00881-7

DO - 10.1016/S0022-2836(03)00881-7

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