title: Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.
abstract: The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.
authors.affiliation: VTT Biotechnical Laboratory, Espoo, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Reinikainen
authors.forename: L
authors.initials: L
authors.lastname: Ruohonen
authors.forename: T
authors.initials: T
authors.lastname: Nevanen
authors.forename: L
authors.initials: L
authors.lastname: Laaksonen
authors.forename: P
authors.initials: P
authors.lastname: Kraulis
authors.forename: T A
authors.initials: TA
authors.lastname: Jones
authors.forename: J K
authors.initials: JK
authors.lastname: Knowles
authors.forename: T T
authors.initials: TT
authors.lastname: Teeri
mesh_terms: D000327:Adsorption; D001665:Binding Sites / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D003460:Crystallization; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D002482:Cellulose; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase