Structure of the cellulosome of Clostridium thermocellum: Role of calcium in assembly of the cellulosome.

摘要

The cellulosome (cellulolytic complex) of Clostridium thermocellum JW20 consists of 26 different polypeptides and contains a significant amount of calcium and phosphorus and a minor amount of iron, zinc and copper. Removal of calcium by EDTA or EGTA resulted in dissociation of the cellulosome. The dissociated cellulosome retains about 30% of the crystalline cellulolytic activity and 100% of the endoglucanase activity compared with the intact cellulosome. The dissociation using EDTA provided a way to obtain and identify individual subunits. Unexpected was the finding that after the EDTA-treatment some of the polypeptides were about 7 kDa smaller than they were in the original cellulosome and these polypeptides had reduced calcium binding properties compared to their native subunits.;Two polypeptides of the cellulosome, the 46 kDa and 71 kDa subunits, were purified from the EDTA-treated cellulosome and characterized. The 46 kDa subunit had a N-terminal sequence and enzymatic properties similar to that of CelA. The 71 kDa subunit was identified as a truncated form of CelS. CelA and CelS are subunits of the cellulosome. C-terminal sequencing showed that the 71 kDa subunit was 60 amino acid residues shorter than CelS and that these amino acids had been removed from the C-terminus. This removal forming the 71 kDa truncated form of CelS was caused by the EDTA-treatment. The reaction required oxygen. The site of cleavage was specific and occurred after Asp-681 of CelS.;Several of the enzymatically active subunits of the cellulosome contain a conserved duplicated region (cdr1 and cdr2) close to the C-terminal ends. The 60 amino acid piece removed from CelS included almost all of cdr1 and all of cdr2. The cdr region binds the catalytic subunits of the cellulosome to CipA, a 198 kDa polypeptide functioning as a scaffold. It contains 9 internally repeated element (IRE) which bind the catalytic subunits.;Both cdr1 and cdr2 were chemically synthesized as branched forms (bcdr1 and bcdr2) and antibodies were raised against them. Major subunits of the EDTA-treated cellulosome (91 kDa, 46 kDa and 71 kDa proteins) reacted with anti-bcdr1 but not with anti-bcdr2. Western blots of the untreated cellulosome with anti-bcdr1 and anti-bcdr2 showed that more than 15 subunits reacted with the two antibodies. The part removed from cellulosomal subunits during dissociation binds calcium. This binding was with cdr1 and not with cdr2. Calcium was required for binding of bcdr1 to CipA. In addition to the CipA, two not yet characterized subunits of 160 kDa and 132 kDa showed calcium dependent interaction with bcdr1, indicating that they like CipA may serve as structural proteins.