Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Abstract

(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.

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

“…2A-C). Thus, cohesin-dockerin binding occurs, and CBM-mediated attachment to cellulose enhances the rate of sugar release from crystalline cellulose, as observed previously [21]. Figure 2D directly compares the activity profiles for the dockerin-containing cellulases in the presence of C. thermocellum miniscaffoldin.…”

Evidence for a dual binding mode of dockerin modules to cohesins

“…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.…”

The Clostridium cellulolyticum Dockerin Displays a Dual Binding Mode for Its Cohesin Partner

“…The cellulosome is assembled by the binding of the catalytic subunits, comprising glycoside hydrolases, esterases, and lyases, to a noncatalytic protein scaffold (hereafter referred to as Cip) (5). The integration of the plant cell wall hydrolases into the cellulosome has been proposed to potentiate the synergistic interactions between the enzymes and contributes to substrate targeting through the cellulose binding capacity of most Cip molecules (6,7). The Cip molecules of Clostridium cellulolyticum and Clostridium thermocellum (designated CipC and CipA, respectively) can bind eight and nine enzymes, respectively (8), although the cellulosomes of other anaerobic bacteria deploy multiple Cip and adapter molecules in assembling as many as 96 catalytic subunits into a single complex (9).…”