TY - JOUR
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.
N1 - Funding Information:
We thank the NSRRC of Taiwan, SPring-8 and the Photon Factory of Japan for beam time. This work was supported by National Cheng Kung University, Academia Sinica, the Ministry of Science and Technology of Taiwan (grant no. NSC 102-2311-B-006-006-MY3 to W.Y.J. and NSC 99-3113-B-001-001 to A.H.J.W.). This work was also partially supported by the Taiwan Protein Project (MOST105-0210-01-12-01 and MOST106-0210-01-15-04) and the Program for Translational Innovation of Biopharmaceutical Development–Technology Supporting Platform Axis.
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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U2 - 10.1002/cbic.201800789
DO - 10.1002/cbic.201800789
M3 - Article
C2 - 30609216
AN - SCOPUS:85060077202
SN - 1439-4227
VL - 20
SP - 295
EP - 307
JO - ChemBioChem
JF - ChemBioChem
IS - 2
ER -