The Fibronectin Type 3-Like Repeat from the Clostridium thermocellum Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface

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Abstract
Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn31,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn31,2 (more efficient), and Gh9-Fn31,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn31,2, and then Gh9-Fn31,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn31,2, or Fn31,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn31,2, Fn31,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn31,2 and Fn31,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 μmoles g−1 and relative affinities (Kr) of 1.12 and 2.13 liters g−1, respectively. Fn31,2 bound weakly to both celluloses. Fn31,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 μmoles g−1 and Krs of 1.14 and 1.98 liters g−1, respectively. Fn31,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn31,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn31,2 on the cellulose surface.