Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability?

Cellulolytic and hemicellulolytic enzymes usually have a domain composition. The mutual influence of a cellulose-binding domain and a catalytic domain was investigated with cellobiohydrolase CelK and xylanase XynZ from Clostridium thermocellum, CelK is composed of an N-terminal family TV cellulose-binding domain (CBDIVCelK), a family 9 glycosyl hydrolase domain (Gh9(CelK)) and a dockerin domain (DD), CelK without the DD, (CBDIV-Gh9)(CelK) and CBDIVCelK bound cellulose. The thermostability of (CBDIV-Gh9)(CelK) was significantly higher than that of CBDIVCelK and Gh9(CelK). The temperature optima of (CBDIV-Gh9)(Celk) and Gh9(CelK) were 65 and 45 degreesC, respectively. XynZ consists of an N-terminal feruloyl esterase domain (FAE(XynZ)), a linker (L), a family VI CBD (CBDVIXynZ), a DD and a xylanase domain, FAE(XynZ) and (FAE-L-CBDVI)(XynZ), used in the present study did not bind cellulose, but both were highly thermostable. Replacement of CBDVIXynZ with CBDIVCelK resulted in chimeras with feruloyl esterase activity and the ability to bind cellulose. CBDIVCelK-FAE(XynZ) bound cellulose with parameters similar to that of (CBDIV-Gh9)(CelK). (FAE-L)(XynZ)-CBDIVCelK and FAE(XynZ)-CBDIVCelK had lower relative affinities and binding capacities than those of (CBDIV-Gh9)(Celk). The three chimeras were much less thermostable than FAE(XynZ) and (FAE-L-CBDVI)(XynZ). The results indicate that domains of glycosyl hydrolases are not randomly combined and that domain interactions affect properties of these domain-structured enzymes.