Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity

To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA(CBD)) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellum endoglucanase (EGE). The two CBDs exhibited similar K-a values for bacterial microcrystalline cellulose (CELECBD, 1.62 x 10(6) M-1; XYLA(CBD), 1.83 x 10(6) M-1) and acid-swollen cellulose (CELECBD, 1.66 x 10(6) M-1; XYLA(CBD), 1.73 x 10(6) M-1). NMR spectra of XYLA(CBD) titrated with cello-oligosaccharides showed that the envicello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The K-a values of the XYLA(CBD) for C-6, C-5 and C-4 cello-oligosaccharides were estimated to be 3.3 x 10(2), 1.4 x 10(2) and 4.0 x 10(1) M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLA(CBD) to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates, Pseudomonas CBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLA(CBD) and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.