Crystal Structures of the C-Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris

Wen Yih Jeng, Chia I. Liu, Te Jung Lu, Hong Jie Lin, Nai Chen Wang, Andrew H.J. Wang

Research output: Contribution to journalArticle

Abstract

Endoglucanase CtCel9Q is one of the enzyme components of the cellulosome, which is an active cellulase system in the thermophile Clostridium thermocellum. The precursor form of CtCel9Q comprises a signal peptide, a glycoside hydrolase family 9 catalytic domain, a type 3c carbohydrate-binding module (CBM), and a type I dockerin domain. Here, we report the crystal structures of C-terminally truncated CtCel9Q (CtCel9QΔc) complexed with Tris, Tris+cellobiose, cellobiose+cellotriose, cellotriose, and cellotetraose at resolutions of 1.50, 1.70, 2.05, 2.05 and 1.75 Å, respectively. CtCel9QΔc forms a V-shaped homodimer through residues Lys529–Glu542 on the type 3c CBM, which pairs two β-strands (β4 and β5 of the CBM). In addition, a disulfide bond was formed between the two Cys535 residues of the protein monomers in the asymmetric unit. The structures allow the identification of four minus (−) subsites and two plus (+) subsites; this is important for further understanding the structural basis of cellulose binding and hydrolysis. In the oligosaccharide-free and cellobiose-bound CtCel9QΔc structures, a Tris molecule was found to be bound to three catalytic residues of CtCel9Q and occupied subsite −1 of the CtCel9Q active-site cleft. Moreover, the enzyme activity assay in the presence of 100 mm Tris showed that the Tris almost completely suppressed CtCel9Q hydrolase activity.

Original languageEnglish
Pages (from-to)295-307
Number of pages13
JournalChemBioChem
Volume20
Issue number2
DOIs
Publication statusPublished - Jan 18 2019

Fingerprint

Clostridium thermocellum
Cellobiose
Clostridium
Cellulase
Crystal structure
Carbohydrates
Catalytic Domain
Cellulosomes
Glycoside Hydrolases
Enzyme Assays
Enzyme activity
Hydrolases
Protein Sorting Signals
Oligosaccharides
Cellulose
Disulfides
Hydrolysis
Assays
Monomers
Molecules

Keywords

  • cellulases
  • cellulosome
  • glycosides
  • hydrolases
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

Cite this

Crystal Structures of the C-Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris. / Jeng, Wen Yih; Liu, Chia I.; Lu, Te Jung; Lin, Hong Jie; Wang, Nai Chen; Wang, Andrew H.J.

In: ChemBioChem, Vol. 20, No. 2, 18.01.2019, p. 295-307.

Research output: Contribution to journalArticle

Jeng, Wen Yih ; Liu, Chia I. ; Lu, Te Jung ; Lin, Hong Jie ; Wang, Nai Chen ; Wang, Andrew H.J. / Crystal Structures of the C-Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris. In: ChemBioChem. 2019 ; Vol. 20, No. 2. pp. 295-307.
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abstract = "Endoglucanase CtCel9Q is one of the enzyme components of the cellulosome, which is an active cellulase system in the thermophile Clostridium thermocellum. The precursor form of CtCel9Q comprises a signal peptide, a glycoside hydrolase family 9 catalytic domain, a type 3c carbohydrate-binding module (CBM), and a type I dockerin domain. Here, we report the crystal structures of C-terminally truncated CtCel9Q (CtCel9QΔc) complexed with Tris, Tris+cellobiose, cellobiose+cellotriose, cellotriose, and cellotetraose at resolutions of 1.50, 1.70, 2.05, 2.05 and 1.75 {\AA}, respectively. CtCel9QΔc forms a V-shaped homodimer through residues Lys529–Glu542 on the type 3c CBM, which pairs two β-strands (β4 and β5 of the CBM). In addition, a disulfide bond was formed between the two Cys535 residues of the protein monomers in the asymmetric unit. The structures allow the identification of four minus (−) subsites and two plus (+) subsites; this is important for further understanding the structural basis of cellulose binding and hydrolysis. In the oligosaccharide-free and cellobiose-bound CtCel9QΔc structures, a Tris molecule was found to be bound to three catalytic residues of CtCel9Q and occupied subsite −1 of the CtCel9Q active-site cleft. Moreover, the enzyme activity assay in the presence of 100 mm Tris showed that the Tris almost completely suppressed CtCel9Q hydrolase activity.",
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T1 - Crystal Structures of the C-Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris

AU - Jeng, Wen Yih

AU - Liu, Chia I.

AU - Lu, Te Jung

AU - Lin, Hong Jie

AU - Wang, Nai Chen

AU - Wang, Andrew H.J.

PY - 2019/1/18

Y1 - 2019/1/18

N2 - Endoglucanase CtCel9Q is one of the enzyme components of the cellulosome, which is an active cellulase system in the thermophile Clostridium thermocellum. The precursor form of CtCel9Q comprises a signal peptide, a glycoside hydrolase family 9 catalytic domain, a type 3c carbohydrate-binding module (CBM), and a type I dockerin domain. Here, we report the crystal structures of C-terminally truncated CtCel9Q (CtCel9QΔc) complexed with Tris, Tris+cellobiose, cellobiose+cellotriose, cellotriose, and cellotetraose at resolutions of 1.50, 1.70, 2.05, 2.05 and 1.75 Å, respectively. CtCel9QΔc forms a V-shaped homodimer through residues Lys529–Glu542 on the type 3c CBM, which pairs two β-strands (β4 and β5 of the CBM). In addition, a disulfide bond was formed between the two Cys535 residues of the protein monomers in the asymmetric unit. The structures allow the identification of four minus (−) subsites and two plus (+) subsites; this is important for further understanding the structural basis of cellulose binding and hydrolysis. In the oligosaccharide-free and cellobiose-bound CtCel9QΔc structures, a Tris molecule was found to be bound to three catalytic residues of CtCel9Q and occupied subsite −1 of the CtCel9Q active-site cleft. Moreover, the enzyme activity assay in the presence of 100 mm Tris showed that the Tris almost completely suppressed CtCel9Q hydrolase activity.

AB - Endoglucanase CtCel9Q is one of the enzyme components of the cellulosome, which is an active cellulase system in the thermophile Clostridium thermocellum. The precursor form of CtCel9Q comprises a signal peptide, a glycoside hydrolase family 9 catalytic domain, a type 3c carbohydrate-binding module (CBM), and a type I dockerin domain. Here, we report the crystal structures of C-terminally truncated CtCel9Q (CtCel9QΔc) complexed with Tris, Tris+cellobiose, cellobiose+cellotriose, cellotriose, and cellotetraose at resolutions of 1.50, 1.70, 2.05, 2.05 and 1.75 Å, respectively. CtCel9QΔc forms a V-shaped homodimer through residues Lys529–Glu542 on the type 3c CBM, which pairs two β-strands (β4 and β5 of the CBM). In addition, a disulfide bond was formed between the two Cys535 residues of the protein monomers in the asymmetric unit. The structures allow the identification of four minus (−) subsites and two plus (+) subsites; this is important for further understanding the structural basis of cellulose binding and hydrolysis. In the oligosaccharide-free and cellobiose-bound CtCel9QΔc structures, a Tris molecule was found to be bound to three catalytic residues of CtCel9Q and occupied subsite −1 of the CtCel9Q active-site cleft. Moreover, the enzyme activity assay in the presence of 100 mm Tris showed that the Tris almost completely suppressed CtCel9Q hydrolase activity.

KW - cellulases

KW - cellulosome

KW - glycosides

KW - hydrolases

KW - X-ray crystallography

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