Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome me diates the tight binding of cellulolytic enzymes to the cellulosome-integra ting protein CipA. Here, this interaction was used to study the effect of d ifferent cellulose-binding domains (CBDs) on the enzymatic activity of C. t hermocellum endoglucanase CelD (1,4-beta-D endoglucanase, EC3.2.1.4) toward various cellulosic substrates. The seventh cohesin domain of CipA was fuse d to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBDCBH1 and CBDCBH2) (1,4-beta-D glucan-cellobiohydrolase. EC3.2.1.91), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBDCex) (beta-1,4-D glucanase, EC3.2.1.8), and from C. thermocellum CipA (CBDCipA). The CBD-coh esin hybrids interacted with the dockerin domain of CelD. leading to the fo rmation of CelD-CBD complexes. Each of the CBDs increased the fraction of c ellulose accessible to hydrolysis by CelD in the order CBDCBH1 < CBDCBH2 ap proximate to CBDCex < CBDCipA. In all cases, the extent of hydrolysis was l imited by the disappearance of sites accessible to CelD. Addition of a batc h of fresh cellulose after completion of the reaction resulted in a new bur st of activity, proving the reversible binding of the intact complexes desp ite the apparent binding irreversibility of some CBDs. Furthermore, burst o f activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation bet ween different CBDs suggests that the sites made available for hydrolysis b y each of the CBDs are at least partially nonoverlapping. The only exceptio n was CBDCipA, whose sites appeared to overlap all of the other sites.