(560c) Dissecting the Cellulosome Enzyme Complex: Assembly and Function
Most bacteria and fungi use free enzymes to degrade plant cell walls. However, a small number of bacteria have developed a fundamentally different approach, where enzymes are tethered to a large protein scaffold forming a multi-domain complex known as, the cellulosome. The study of these large protein assemblies is an ongoing research topic that has already yielded numerous breakthroughs. However, the mechanism of assembly of the enzyme subunits onto the natural scaffoldin; as well as the modes of action of the cellulosome and its enzymatic components, are currently not well understood.
In this study, we focused on the cellulosome-integrating protein (CipA) of C. thermocellum; as well as cellulosomal enzymes from families 5, 9 and 48. These three enzymes are representative of the diversity of enzymes secreted by C. thermocellum. This work not only aims at understanding the mechanisms involved in the sequential binding of the cellulosomal enzymes to the CipA scaffold of C. thermocellum, but also the binding of CipA to secondary scaffolds, such as OlpB. We focused on the physical properties of the binding of component enzymes to the scaffoldin. The modularity of the enzymes was found to be one of the main influences on the cellulosome assembly process.
Additionally, we examined several modules from the multi-modular enzyme complex, CbhA. Several of these modules have undefined functions, such as the X1 modules found closely linked to the catalytic domain. We hypothesize that the X modules could serve as tethers between several modules in CbhA to fine-tune the positioning of this enzyme during cellulose deconstruction. We conducted steered molecular dynamics simulations (directly comparable to AFM pulling experiments) to evaluate the energy and the profile for unfolding these modules. We found that the energy required to partially unfold this domain is accessible by the cellulosome and the unfolding pathways are similar in the multiple simulations conducted.