Synergy, structure and conformational flexibility of hybrid cellulosomes displaying various inter-cohesins linkers.
Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.
Molinier
Anne-Laure
AL
LCB-CNRS UPR9043 IMM, 31, chemin Joseph-Aiguier, 13402, Marseille cedex 20, France.
Nouailler
Matthieu
M
Valette
Odile
O
Tardif
Chantal
C
Receveur-Bréchot
Véronique
V
Fierobe
Henri-Pierre
HP
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology; D048013:Acetivibrio thermocellus / Q000201:enzymology; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle
D002352:Carrier Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002482:Cellulose