The fibronectin type 3-like repeat from the Clostridium thermocellum cellobiohydrolase CbhA promotes hydrolysis of cellulose by modifying its surface.
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 (Fn3(1,2)), 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-Fn3(1,2) (more efficient), and Gh9-Fn3(1,2)-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn3(1,2), and then Gh9-Fn3(1,2)-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn3(1,2,) or Fn3(1,2)-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn3(1,2), Fn3(1,2)-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn3(1,2) and Fn3(1,2)-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 micro moles g(-1) and relative affinities (K(r)) of 1.12 and 2.13 liters g(-1), respectively. Fn3(1,2) bound weakly to both celluloses. Fn3(1,2)-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 micro moles g(-1) and K(r)s of 1.14 and 1.98 liters g(-1), respectively. Fn3(1,2) and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn3(1,2) aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn3(1,2) on the cellulose surface.
Kataeva
Irina A
IA
Department of Biochemistry and Molecular Biology and Center for Biological Resources Recovery, The University of Georgia, Athens, Georgia 30602-7229, USA. kataeva@arches.uga.edu
Seidel
Ronald D
RD
Shah
Ashit
A
West
Larry T
LT
Li
Xin-Liang
XL
Ljungdahl
Lars G
LG
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology* / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D005353:Fibronectins; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D002118:Calcium