Multidomain Structure and Cellulosomal Localization of the Clostridium thermocellum Cellobiohydrolase CbhA |
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| Authors: | Vladimir V Zverlov Galina V Velikodvorskaya Wolfgang H Schwarz Karin Bronnenmeier Josef Kellermann and Walter L Staudenbauer |
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| Affiliation: | Institute of Molecular Genetics, Russian Academy of Science, 123182 Moscow, Russia,1. and Institute for Microbiology, Technical University Munich, 80290 Munich,2. and Max-Planck-Institute for Biochemistry, 82152 Martinsried,3. Germany |
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| Abstract: | The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria. |
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