scholarly journals Properties and Mutation Analysis of the CelK Cellulose-Binding Domain from the Clostridium thermocellum Cellulosome

2001 ◽  
Vol 183 (5) ◽  
pp. 1552-1559 ◽  
Author(s):  
Irina A. Kataeva ◽  
Ronald D. Seidel ◽  
Xin-Liang Li ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Calcium Binding ◽  
Clostridium Thermocellum ◽  
Binding Capacity ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Calcium Binding Site ◽  
Aromatic Residues ◽  
Cellulose Binding ◽  
Family Iv

ABSTRACT The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBDCelK) was expressed inEscherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 μmol/g of cellulose and relative affinities (K r) of 2.33 and 9.87 liters/g, respectively. The CBDCelK is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBDCelKalso binds to the soluble polysaccharides lichenin, glucomannan, and barley β-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBDCelK contains 1 mol of calcium per mol. The CBDCelK has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBDCelK four highly conserved aromatic residues (Trp56, Trp94, Tyr111, and Tyr136) and Asp192 were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-NCelK. The CBD-NCelK and D192A retained binding parameters close to that of the intact CBDCelK, W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K rand Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBDCelK led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBDCelK and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBDCelK are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBDN1Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBDCelK correctly folded.

scholarly journals The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain

1991 ◽  
Vol 273 (2) ◽  
pp. 289-293 ◽  
Author(s):  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Amino Acids ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Full Length ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Beta Glucan ◽  
Cellulose Binding

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

scholarly journals Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes

1997 ◽  
Vol 326 (2) ◽  
pp. 617-624 ◽  
Author(s):  
Irina KATAEVA ◽  
Gérard GUGLIELMI ◽  
Pierre BÉGUIN
Keyword(s):  
Clostridium Thermocellum ◽  
Analytical Ultracentrifugation ◽  
Cellulase Activity ◽  
Hydrolytic Activity ◽  
Binding Domain ◽  
Cellulolytic Activity ◽  
Cellulose Binding Domain ◽  
Optimal Ratio ◽  
Cellulose Binding

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD–CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.

Crystallization and Preliminary X-ray Analysis of the Major Cellulose-binding Domain of the Cellulosome from Clostridium thermocellum

1994 ◽  
Vol 244 (2) ◽  
pp. 236-237 ◽  
Author(s):  
Raphael Lamed ◽  
José Tormo ◽  
Arthur J. Chirino ◽  
Ely Morag ◽  
Edward A. Bayer
Keyword(s):  
Clostridium Thermocellum ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
X Ray ◽  
Cellulose Binding

scholarly journals Characterization of EngF from Clostridium cellulovorans and Identification of a Novel Cellulose Binding Domain

1998 ◽  
Vol 64 (3) ◽  
pp. 1086-1090 ◽  
Author(s):  
Akihiko Ichi-ishi ◽  
Salah Sheweita ◽  
Roy H. Doi
Keyword(s):  
Amino Acids ◽  
Cellulose Degradation ◽  
Binding Activity ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Binding Studies ◽  
Endoglucanase Activity ◽  
Clostridium Cellulovorans ◽  
C Terminus ◽  
Cellulose Binding

ABSTRACT The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied. Binding studies revealed that the Kd and the maximum amount of protein bound for acid-swollen cellulose were 1.8 μM and 7.1 μmol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.

scholarly journals Identification of functionally important amino acids in the cellulose-binding domain ofTrichoderma reeseicellobiohydrolase I

1995 ◽  
Vol 4 (6) ◽  
pp. 1056-1064 ◽  
Author(s):  
Markus Linder ◽  
Maija-Liisa Mattinen ◽  
Maarit Kontteli ◽  
Gunnar Lindeberg ◽  
Jerry Ståhlberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Cellulose Binding

Calcium Binding by the N-Terminal Cellulose-Binding Domain fromCellulomonasfimiβ-1,4-Glucanase CenC†

Biochemistry ◽  
1998 ◽  
Vol 37 (37) ◽  
pp. 12772-12781 ◽  
Author(s):  
Philip E. Johnson ◽  
A. Louise Creagh ◽  
Emmanuel Brun ◽  
Koman Joe ◽  
Peter Tomme ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Cellulose Binding

scholarly journals Involvement of Both Dockerin Subdomains in Assembly of the Clostridium thermocellum Cellulosome

1998 ◽  
Vol 180 (24) ◽  
pp. 6581-6585 ◽  
Author(s):  
Betsy Lytle ◽  
J. H. David Wu
Keyword(s):  
Calcium Binding ◽  
Clostridium Thermocellum ◽  
Scaffolding Protein ◽  
Stable Complex ◽  
Binding Motif ◽  
Cellulose Binding Domain ◽  
Catalytic Subunits ◽  
The Third ◽  
Ef Hand ◽  
Cellulose Binding

ABSTRACT Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.

Sign in / Sign up

Export Citation Format

Share Document