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.

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

“…The second tryptophan to be oxidized is in the catalytic domain (Macarron et al, 1995). Site-directed mutation supports the conclusion that exposed aromatic amino acids of family I CBDs are important in binding to cellulose (Reinikainen et al, 1992). Thus, oxidation of the tryptophans in CBDcex with NBS could be used to analyze in some detail the interaction of this CBD with cellulose.…”

The difference in affinity between two fungal cellulose‐binding domains is dominated by a single amino acid substitution

“…Those amino acids which were investigated in this study are marked with boxes. The numbering used is based on the CBHI-CBD for clarity when comparing with other work [3,6,7].…”

“…Indeed, our preliminary data obtained with a hybrid CBHI with the EGI CBD instead of its own, revealed improved binding but no changes in the enzymatic activity of CBHI [9]. On the other hand, mutations decreasing the affinity of the CBHI CBD do decrease its catalytic activity on crystalline cellulose [3,4]. Therefore, it is possible that beyond a certain, relatively high affinity of the CBD slight increases will no longer improve the enzymatic activity on crystalline cellulose.…”

“…The CBDs of fungal enzymes are very similar in their amino acid sequence and size [1]. These small domains are responsible for most of the enzyme's affinity for cellulose [2][3][4][5]. The cellulose binding mediated by the CBD is essential for the enzymatic activity on crystalline cellulose, but has no effect on the hydrolysis of soluble substrates.…”

“…This is the major cellulolytic enzyme of the fungus, and it is capable of efficient hydrolysis of crystalline cellulose. The three-dimensional structure of the CBHI CBD has been solved [6], permitting detailed structure-function studies [3,4,7]. So far three tyrosines, one glutamine and one asparagine have been proposed to participate in the binding.…”