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

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Abstract
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 IV cellulose-binding domain (CBDIVCelK), a family 9 glycosyl hydrolase domain (Gh9CelK) 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 Gh9CelK. The temperature optima of (CBDIV–Gh9)CelK and Gh9CelK were 65 and 45°C, respectively. XynZ consists of an N-terminal feruloyl esterase domain (FAEXynZ), a linker (L), a family VI CBD (CBDVIXynZ), a DD and a xylanase domain. FAEXynZ 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–FAEXynZ bound cellulose with parameters similar to that of (CBDIV–Gh9)CelK. (FAE–L)XynZ–CBDIVCelK and FAEXynZ–CBDIVCelK had lower relative affinities and binding capacities than those of (CBDIV–Gh9)CelK. The three chimeras were much less thermostable than FAEXynZ 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.