Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.
The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation.
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Bioénérgetique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM-IFR1, Marseille, France.
Gal
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Bélaïch
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Gaudin
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Tardif
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Bélaïch
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JP
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
D001426:Bacterial Proteins; D002352:Carrier Proteins; C098847:CipC protein, Clostridium cellulolyticum; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C074775:CelA endoglucanase; D002480:Cellulase