Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.
Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.
Linder
M
M
Department of Biochemistry, University of Uppsala, Sweden.
Mattinen
M L
ML
Kontteli
M
M
Lindeberg
G
G
Ståhlberg
J
J
Drakenberg
T
T
Reinikainen
T
T
Pettersson
G
G
Annila
A
A
D000327:Adsorption; D000595:Amino Acid Sequence; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D017124:Conserved Sequence; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014242:Trichoderma / Q000201:enzymology*
D010446:Peptide Fragments; D002482:Cellulose; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase