Design and Production of Active Cellulosome Chimeras

Fierobe, Henri‐Pierre; Mechaly, Adva; Tardif, Chantal; Belaı̈ch, Anne; Lamed, Raphael; Shoham, Yuval; Bélaı̈ch, Jean-Pierre; Bayer, Edward A.

doi:10.1074/jbc.m102082200

Cited by 188 publications

(99 citation statements)

References 32 publications

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“…3B, c). As above, these results can be explained on the basis of the inferior affinity characteristics for the cohesin-dockerin interaction of C. cellulolyticum versus those of C. thermocellum (21,22). Despite the relatively modest affinity observed for these chimeras, the findings also support the premise that in C. cellulolyticum, the relevant region resides within the second and last sixth of the protein.…”

Section: Resultssupporting

confidence: 72%

“…The preference in favor of the C. thermocellum system can be explained by the superior affinity characteristics for the cohe- sin-dockerin interaction of the latter over those of C. cellulolyticum, as observed previously (5,15,(21)(22)(23). In any case, Sw1-2, which contains both the first and last third of the C. thermocellum cohesin, binds strongest to the corresponding dockerin.…”

Section: Resultsmentioning

confidence: 56%

“…The very high affinity of both cohesins (K D Ͻ 10 Ϫ10 M) to their matching dockerins (15,(21)(22)(23) suggests that numerous intermodular interactions occur between the cohesin and dockerin. On the other hand, we reasoned that not all of the interactions would be necessary for interspecies specificity.…”

mentioning

confidence: 99%

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Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping

Nakar

Handelsman

Shoham

et al. 2004

Journal of Biological Chemistry

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The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with ϳ20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 56%

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Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping

Nakar

Handelsman

Shoham

et al. 2004

Journal of Biological Chemistry

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“…This flexibility in the arrangement of catalytic subunits within cellulosomes is important as different (and potentially temporally evolving) enzyme combinations are required to degrade the myriad of composite structures displayed by plant cell walls (1). Indeed, the incorporation of complementary enzyme activities into single cellulosomes potentiates the synergistic interactions between these biocatalysts and is a key element for the optimization of plant cell wall degradation (33)(34)(35). In addition, the switching of the mode of binding from one site to another can introduce quaternary flexibility into the multienzyme complex and enhance substrate targeting and hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Evidence for a dual binding mode of dockerin modules to cohesins

Carvalho

Dias²,

Nagy

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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129

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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 o 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.cellulosome structure ͉ cellulosome assembly ͉ Clostridium thermocellum ͉ cohesin-dockerin

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“…In previous studies (6,11), the property of species specificity was indeed exploited to selectively incorporate desired enzymes into precise positions within chimeric minicellulosomes. In this approach, chimeric scaffoldins were constructed that contained a cohesin from each species and an optional CBM.…”

mentioning

confidence: 99%

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Fierobe

Mingardon

Mechaly

et al. 2005

Journal of Biological Chemistry

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217

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(2002) J. Biol. Chem. 277, 49621-49630), 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.

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Design and Production of Active Cellulosome Chimeras

Cited by 188 publications

References 32 publications

Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping

Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping

Evidence for a dual binding mode of dockerin modules to cohesins

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

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