Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei

Jin-yu Zou, Gerald Kleywegt, Jerry Ståhlberg, Hugues Driguez, Wim Nerinckx, Marc Claeyssens, Anu Koivula, Tuula Teeri, Alwyn Jones (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.

Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc)2-S-(Glc)2, which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG).

Conclusions: The (Glc)2-S-(Glc)2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)2-S-(Glc)2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.
Original languageEnglish
Pages (from-to)1035-1045
Number of pages11
JournalStructure
Volume7
Issue number9
DOIs
Publication statusPublished - 1999
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Catalysis
Ligands
Catalytic Domain
Proteins
Enzymes
Oligosaccharides
Sulfur
Oxygen

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Zou, Jin-yu ; Kleywegt, Gerald ; Ståhlberg, Jerry ; Driguez, Hugues ; Nerinckx, Wim ; Claeyssens, Marc ; Koivula, Anu ; Teeri, Tuula ; Jones, Alwyn. / Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei. In: Structure. 1999 ; Vol. 7, No. 9. pp. 1035-1045.
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title = "Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei",
abstract = "Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc)2-S-(Glc)2, which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG).Conclusions: The (Glc)2-S-(Glc)2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)2-S-(Glc)2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.",
author = "Jin-yu Zou and Gerald Kleywegt and Jerry St{\aa}hlberg and Hugues Driguez and Wim Nerinckx and Marc Claeyssens and Anu Koivula and Tuula Teeri and Alwyn Jones",
year = "1999",
doi = "10.1016/S0969-2126(99)80171-3",
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Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei. / Zou, Jin-yu; Kleywegt, Gerald; Ståhlberg, Jerry; Driguez, Hugues; Nerinckx, Wim; Claeyssens, Marc; Koivula, Anu; Teeri, Tuula; Jones, Alwyn (Corresponding Author).

In: Structure, Vol. 7, No. 9, 1999, p. 1035-1045.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei

AU - Zou, Jin-yu

AU - Kleywegt, Gerald

AU - Ståhlberg, Jerry

AU - Driguez, Hugues

AU - Nerinckx, Wim

AU - Claeyssens, Marc

AU - Koivula, Anu

AU - Teeri, Tuula

AU - Jones, Alwyn

PY - 1999

Y1 - 1999

N2 - Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc)2-S-(Glc)2, which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG).Conclusions: The (Glc)2-S-(Glc)2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)2-S-(Glc)2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

AB - Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc)2-S-(Glc)2, which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG).Conclusions: The (Glc)2-S-(Glc)2 ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)2-S-(Glc)2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

U2 - 10.1016/S0969-2126(99)80171-3

DO - 10.1016/S0969-2126(99)80171-3

M3 - Article

VL - 7

SP - 1035

EP - 1045

JO - Structure

JF - Structure

SN - 0969-2126

IS - 9

ER -