MULTIDOMAIN STRUCTURE AND CELLULOSOMAL LOCALIZATION OF THE CLOSTRIDIUM-THERMOCELLUM CELLOBIOHYDROLASE CBHA

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open re ading frame encoding a protein of 1,230 amino acids was identified. Re moval of a putative signal peptide yields a mature protein of 1,203 am ino acids with a molecular weight of 135,139. Sequence analysis of Cbh A reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family TV, an immunoglobulin-like P-barrel domain, a catalytic domain homolog ous to cellulase family El, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acid ic linker region, and a C-terminal dockerin domain. The cellulosomal l ocalization of CbhA was confirmed by Western blot analysis employing p olyclonal antibodies raised against a truncated enzymatically active v ersion of CbhA, CbhA was identified as cellulosomal subunit S3 by part ial amino acid sequence analysis. Comparison of the multidomain struct ures indicates striking similarities between CbhA and a group of cellu lases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spr ead by horizontal gene transfer among gram-positive cellulolytic bacte ria.