Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation.
(17/63)
The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon "intercellular complementation." Interesting implications concerning bacterial cooperation are suggested from these results. (+info)
Evidence for a dual binding mode of dockerin modules to cohesins.
(18/63)
The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived "type I dockerin" modules with the multiple "cohesin" modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180 degrees relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex. (+info)
Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes.
(19/63)
Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes. (+info)
Exploration of new geometries in cellulosome-like chimeras.
(20/63)
In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a "covalent cellulosome" that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency. (+info)
Effect of multiple copies of cohesins on cellulase and hemicellulase activities of Clostridium cellulovorans mini-cellulosomes.
(21/63)
Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpA1), two cohesins (mini-CbpA12), or four cohesins (mini-CbpA1234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and corn fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with corn fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpA1234 (1.8-fold) and then mini-CbpA12 (1.3-fold), and the lowest synergy was observed with mini-CbpA1 (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls. (+info)
Mutations in the scaffoldin gene, cipA, of Clostridium thermocellum with impaired cellulosome formation and cellulose hydrolysis: insertions of a new transposable element, IS1447, and implications for cellulase synergism on crystalline cellulose.
(22/63)