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

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Abstract
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-β- d endoglucanase, EC 3.2.1.4 ) 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-β- d glucan-cellobiohydrolase, EC 3.2.1.91 ), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD Cex ) (β-1,4- d glucanase, EC 3.2.1.8 ), 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 ≈ 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.