NDLI: Properties and Mutation Analysis of the CelK Cellulose-Binding Domain from the Clostridium thermocellum Cellulosome

Content Provider	PubMed Central
Author	Kataeva, Irina A. Seidel, Ronald D. Li, Xin-liang Ljungdahl, Lars G.
Copyright Year	2001
Abstract	The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBDCelK) was expressed in Escherichia 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 CBDCelK also 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 r and 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 CBDN1 Y85, 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.
Related Links	http://dx.doi.org/10.1128/JB.183.5.1552-1559.2001
Starting Page	1552
File Format	PDF
ISSN	10985530
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	5
Volume Number	183
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-03-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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