In:
Journal of Bacteriology, American Society for Microbiology, Vol. 183, No. 5 ( 2001-03), p. 1552-1559
Abstract:
The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD CelK ) 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 CBD CelK is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD CelK 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 CBD CelK contains 1 mol of calcium per mol. The CBD CelK 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 CBD CelK four highly conserved aromatic residues (Trp 56 , Trp 94 , Tyr 111 , and Tyr 136 ) and Asp 192 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-N CelK . The CBD-N CelK and D192A retained binding parameters close to that of the intact CBD CelK , 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 CBD CelK 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 CBD CelK and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD CelK are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD N1 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 CBD CelK correctly folded.
Type of Medium:
Online Resource
ISSN:
0021-9193
,
1098-5530
DOI:
10.1128/JB.183.5.1552-1559.2001
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2001
detail.hit.zdb_id:
1481988-0
SSG:
12
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