Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.
The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.
McLean
B W
BW
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3.
Bray
M R
MR
Boraston
A B
AB
Gilkes
N R
NR
Haynes
C A
CA
Kilburn
D G
DG
D000192:Actinomycetales / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002482:Cellulose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; C109691:microcrystalline cellulose