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Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

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 beta-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.

SUBMITTER: Zverlov VV

PROVIDER: S-EPMC107808 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

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Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

Zverlov V V VV Velikodvorskaya G V GV Schwarz W H WH Bronnenmeier K K Kellermann J J Staudenbauer W L WL

Journal of bacteriology 19980601 12

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 imm ...[more]

PMID: 9620957

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Project description:BackgroundClostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.ResultsWe constructed C. thermocellum mutants with the primary scaffoldin CipA (cellulosome-integrating protein A) truncated at different positions or lacking four different secondary scaffoldins by using a newly developed thermotargetron system, and we analyzed cellulose hydrolysis, cellulosome formation, and cellulose binding of the mutants. A CipA truncation that deletes six type I cohesin modules, which bind cellulolytic enzymes, decreased cellulose hydrolysis rates by 46%, and slightly longer truncations that also delete the carbohydrate binding module decreased rates by 89 to 92%, indicating strong cellulosome-substrate synergy. By contrast, a small CipA truncation that deletes only the C-terminal type II dockerin (XDocII) module detached cellulosomes from the cells, but decreased cellulose hydrolysis rates by only 9%, suggesting a relatively small contribution of cellulosome-cell synergy. Disruptants lacking any of four different secondary scaffoldins (OlpB, 7CohII, Orf2p, or SdbA) showed moderately decreased cellulose hydrolysis rates, suggesting additive contributions. Surprisingly, the CipA-ΔXDocII mutant, which lacks cell-associated polycellulosomes, adheres to cellulose almost as strongly as wild-type cells, revealing an alternate, previously unknown cellulose-binding mechanism.ConclusionsOur results emphasize the important role of cellulosome-substrate synergy in cellulose degradation, demonstrate a contribution of secondary scaffoldins, and suggest a previously unknown, non-cellulosomal system for binding insoluble cellulose. Our findings provide new insights into cellulosome function and impact genetic engineering of microorganisms to enhance bioconversions of cellulose substrates.

Project description:BACKGROUND:Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. RESULTS:Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. CONCLUSIONS:The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Dataset Information

Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

Publications

Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

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