Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from , and .

BACKGROUND

The ascomycete fungus is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) Cel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from (Cel7A) and (Cel7A) show high sequence identity with Cel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.

RESULTS

The Cel7A, Cel7A, and Cel7A enzymes were characterized for comparison of function. The catalytic domain of Cel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with Cel7A than either Cel7A or Cel7A. In synergistic saccharification of pretreated corn stover, both Cel7A and Cel7A were more efficient than Cel7A, although Cel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of spp. GH7 CBHs.

CONCLUSIONS

We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from Cel7A with the thermostability features of Cel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.