title: Degradation of cellulose substrates by cellulosome chimeras. Substrate targeting versus proximity of enzyme components.
abstract: A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Bélaich, A., Lamed, R., Shoham, Y., Bélaich, J.-P., and Bayer, E. A. (2001) J. Biol. Chem. 276, 21257-21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.
authors.affiliation: Bioénergétique et Ingéniérie des Protéines, CNRS, IBSM, 13402 Marseille, France. hpfierob@ibsm.cnrs-mrs.fr
authors.forename: Henri-Pierre
authors.initials: HP
authors.lastname: Fierobe
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.forename: Chantal
authors.initials: C
authors.lastname: Tardif
authors.forename: Mirjam
authors.initials: M
authors.lastname: Czjzek
authors.forename: Adva
authors.initials: A
authors.lastname: Mechaly
authors.forename: Anne
authors.initials: A
authors.lastname: Bélaïch
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Bélaïch
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D003013:Clostridium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D001617:beta-Glucosidase