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

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C. thermocellum CipA (CBD(CipA)). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA). In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites.

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