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, Christophe Braet, Harry Brumer III, Marc Claeyssens, Christina Divne, Richard Fagerström, Mark Harris, Alwyn Jones, Gerald Kleywegt, Anu Koivula, Sabah Mahdi, Kathleen Piens, Michael Sinnott (Corresponding Author), Jerry Ståhlberg, Tuula Teeri, Melanie Underwood, Gerd Wohlfahrt

Research output: Contribution to journalArticleScientificpeer-review

51 Citations (Scopus)

Abstract

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 (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) 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 k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) 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.
Original languageEnglish
Pages (from-to)19-30
Number of pages12
JournalBiochemical Journal
Volume356
Issue number1
DOIs
Publication statusPublished - 2001
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Glycoside Hydrolases
Histidine
Cellobiose
Acids
Cellulase
X ray crystallography
X Ray Crystallography
Enzymes
Ionization
Hydrogen
Hydrolysis
Catalytic Domain
Hydrogen bonds
Crystal structure
Mutation
Catalysts
Water
Substrates

Cite this

Becker, Dieter ; Braet, Christophe ; Brumer III, Harry ; Claeyssens, Marc ; Divne, Christina ; Fagerström, Richard ; Harris, Mark ; Jones, Alwyn ; Kleywegt, Gerald ; Koivula, Anu ; Mahdi, Sabah ; Piens, Kathleen ; Sinnott, Michael ; Ståhlberg, Jerry ; Teeri, Tuula ; Underwood, Melanie ; Wohlfahrt, Gerd. / 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. In: Biochemical Journal. 2001 ; Vol. 356, No. 1. pp. 19-30.
@article{9be929d166544777a4b3bce7dfc73b46,
title = "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",
abstract = "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 (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) 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 k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) 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.",
author = "Dieter Becker and Christophe Braet and {Brumer III}, Harry and Marc Claeyssens and Christina Divne and Richard Fagerstr{\"o}m and Mark Harris and Alwyn Jones and Gerald Kleywegt and Anu Koivula and Sabah Mahdi and Kathleen Piens and Michael Sinnott and Jerry St{\aa}hlberg and Tuula Teeri and Melanie Underwood and Gerd Wohlfahrt",
year = "2001",
doi = "10.1042/0264-6021:3560019",
language = "English",
volume = "356",
pages = "19--30",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland press",
number = "1",

}

Becker, D, Braet, C, Brumer III, H, Claeyssens, M, Divne, C, Fagerström, R, Harris, M, Jones, A, Kleywegt, G, Koivula, A, Mahdi, S, Piens, K, Sinnott, M, Ståhlberg, J, Teeri, T, Underwood, M & Wohlfahrt, G 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', Biochemical Journal, vol. 356, no. 1, pp. 19-30. https://doi.org/10.1042/0264-6021:3560019

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. / Becker, Dieter; Braet, Christophe; Brumer III, Harry; Claeyssens, Marc; Divne, Christina; Fagerström, Richard; Harris, Mark; Jones, Alwyn; Kleywegt, Gerald; Koivula, Anu; Mahdi, Sabah; Piens, Kathleen; Sinnott, Michael (Corresponding Author); Ståhlberg, Jerry; Teeri, Tuula; Underwood, Melanie; Wohlfahrt, Gerd.

In: Biochemical Journal, Vol. 356, No. 1, 2001, p. 19-30.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum

T2 - The pH behaviour of Trichoderma reesei Cel7A and its E223S/A224H/L225V/T226A/D262G mutant

AU - Becker, Dieter

AU - Braet, Christophe

AU - Brumer III, Harry

AU - Claeyssens, Marc

AU - Divne, Christina

AU - Fagerström, Richard

AU - Harris, Mark

AU - Jones, Alwyn

AU - Kleywegt, Gerald

AU - Koivula, Anu

AU - Mahdi, Sabah

AU - Piens, Kathleen

AU - Sinnott, Michael

AU - Ståhlberg, Jerry

AU - Teeri, Tuula

AU - Underwood, Melanie

AU - Wohlfahrt, Gerd

PY - 2001

Y1 - 2001

N2 - 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 (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) 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 k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) 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.

AB - 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 (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) 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 k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) 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.

U2 - 10.1042/0264-6021:3560019

DO - 10.1042/0264-6021:3560019

M3 - Article

VL - 356

SP - 19

EP - 30

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -