Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.
In recent work, we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.
Fierobe
Henri-Pierre
HP
Bioénergétique et Ingénierie des Protéines, CNRS, Institut de Biologie Structurale et Microbiologie, Marseille 13402, France. hpfierob@ibsm.cnrs-mrs.fr
Mingardon
Florence
F
Mechaly
Adva
A
Bélaïch
Anne
A
Rincon
Marco T
MT
Pagès
Sandrine
S
Lamed
Raphael
R
Tardif
Chantal
C
Bélaïch
Jean-Pierre
JP
Bayer
Edward A
EA
D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000201:enzymology* / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D005656:Fungal Proteins; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000097722:Cohesins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002482:Cellulose; D002868:Chromosomal Proteins, Non-Histone; D043364:Endo-1,4-beta Xylanases; D005656:Fungal Proteins; D009687:Nuclear Proteins; D000097722:Cohesins; C074074:cellulose binding protein A, Clostridium cellulovorans