Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5

The upstream region of the cellobiohydrolase gene cbhA of Clostridium therm ocellum F7 was sequenced. It was found that this region contains the previo usly sequenced gene celK encoding an enzyme closely related to CbhA (cellul osomal subunit S3). The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscr ibed as an operon. Sequence comparison between the two cellobiohydrolases r evealed high sequence conservation in the catalytic domain and in the N-ter minal cellulose-binding domain (CBD) homologous to CBD family IV, which bin ds specifically to amorphous cellulose and soluble cellooligosaccharides. I n contrast to CbhA, CelK lacks a family III CBD capable of binding to cryst alline cellulose. By partial amino acid sequence determination CelK was sho wn 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 s tructure, revealed a significant variation in the lengths of substrate-bind ing loops connecting the helices of the (alpha/alpha)(6) barrel fold. The e xtended loops of CelK and CbhA might form an active-site tunnel, as found i n the catalytic domains of fungal cellobiohydrolases.