Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: Evidence for gene duplication and recombination

The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. me report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gen e has an open reading frame (ORF) of 2,685 nucleotides coding for a polypep tide of 895 amino acid residues with a calculated mass of 100,552 Da. A sig nal peptide of 27 amino acid residues is cut off during secretion, resultin g in a mature enzyme of 97,572 Da. The nucleotide sequence is highly simila r to that of cbhA (V.V. Zverlov et al., J. Bacteriol. 180:3091-3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid le vels, respectively. Both have domain structures consisting of a signal pept ide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrol ase domain, and a dockerin domain. A striking distinction between the two p olypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-l ike domain and family III CBD. This insertion, missing in CelK, is responsi ble for the size difference between CelK and CbhA Upstream and downstream f lanking sequences of the two genes show no homology. The data indicate that celK and cbhA in the genome of C. thermocellum have evolved through gene d uplication and recombination of domain coding sequences. celK without a doc kerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65 degrees C, respectively. It hydrol yzed p-nitrophenyl-beta-D-cellobioside with a K-m and a V-max of 1.67 mu M and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK acti vity, with a K-i of 0.29 mM. The enzyme was thermostable, after 200 h of in cubation at 60 degrees C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.