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 xylanas e A (XYLA(CBD)) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocell um 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) titrat ed with cello-oligosaccharides showed that the envicello-oligosacchari des showed that the environment of three tryptophan residues was affec ted when the CBD bound cellohexaose, cellopentaose or cellotetraose. T he K-a values of the XYLA(CBD) for C-6, C-5 and C-4 cello-oligosacchar ides 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 gl ucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLA(C BD) to the catalytic domain of EGE potentiated the activity of the enz yme against insoluble forms of cellulose but not against carboxymethyl cellulose. The increase in cellulase activity was not observed when th e CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respec tively as the substrates, Pseudomonas CBDs did not induce the extensio n of isolated plant cell walls nor weaken cellulose paper strips in th e same way as a class of plant cell wall proteins called expansins. Th e XYLA(CBD) and CELECBD did not release small particles from the surfa ce of cotton. The significance of these results in relation to the mod e of action of Type II CBDs is discussed.