Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5.

The upstream region of the cellobiohydrolase gene cbhA of Clostridium thermocellum F7 was sequenced. It was found that this region contains the previously sequenced gene celK encoding an enzyme closely related to CbhA (cellulosomal subunit S3). The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscribed as an operon. Sequence comparison between the two cellobiohydrolases revealed high sequence conservation in the catalytic domain and in the N-terminal cellulose-binding domain (CBD) homologous to CBD family IV, which binds specifically to amorphous cellulose and soluble cellooligosaccharides. In contrast to CbhA, CelK lacks a family III CBD capable of binding to crystalline cellulose. By partial amino acid sequence determination CelK was shown to be identical to cellulosomal subunit S5. CelK and CbhA were found to be members of subfamily E1 of cellulase family E (glycosylhydrolase family 9). Sequence comparison of catalytic domains of family E1 cellulases with C. thermocellum CelD, a family E1 endoglucanase of known three-dimensional structure, revealed a significant variation in the lengths of substrate-binding loops connecting the helices of the (alpha/alpha)6 barrel fold. The extended loops of CelK and CbhA might form an active-site tunnel, as found in the catalytic domains of fungal cellobiohydrolases.