Ligand discovery and structural-functional analysis of proteins involved in plant cell wall degradation

The plant cell wall is constituted by recalcitrant polysaccharides with diverse sequences that comprise an abundant source of terrestrial biomass. To efficiently degrade plant cell wall polysaccharides, some cellulolytic bacterial organisms, such as Clostridium thermocellum and Ruminococcus flavefaciens, have an extracellular multi-enzyme complex with catalytic and non-catalytic carbohydrate-binding modules (CBMs). CBMs play a crucial role in enhancing the catalytic efficiency of the enzymes by proximity effect, cell attachment or targeting and disruptive function. The Carbohydrate Active enZymes database (CAZy) organizes the identified CBMs by sequence similarity into different families. Deposition of CBM sequences in the CAZy database is continually growing for which characterization and structure-function analysis is required. In this study we aim to characterize the carbohydrate ligand specificities of C. thermocellum ATCC 27405 and R. flavefaciens FD-1 CBMs assigned to different families in the CAZy database. We performed carbohydrate microarray screening analysis for ligand discovery and crystallization screenings aiming to solve the 3D structures of the CBM-ligand complexes by X-ray crystallography. To complement the information provided by these methodologies we also performed ITC (Isothermal Titration Calorimetry), MST (Microscale Thermophoresis) and affinity gel electrophoresis. With the implementation of this approach it was possible to elucidate different carbohydrate binding specificities for biotechnologically relevant CBMs. The results from the initial carbohydrate microarray screening constitute a functional start point to target CBMs for structural-functional analysis of carbohydrate-recognition. C. thermocellum family 50 (CtCBM50) reveals to be a novel chitin binding LysM domain and binding with insoluble chitin and a β-(1-4)-GlcNAc chitin oligosaccharide was identified. R. flavefaciens FD-1 family 62 CBM (RfCBM62) reveals to be highly specific for a pectic polysaccharide for which the structure is being investigated and binding to galacturonan DP4 was observed. In the scope of this thesis, and as the structural characterization was not achieved in due time, the sequence similarity to known structures inspired the attempt to computationally produce similarity models for the two CBMs. The (hypothetical) conservation of the secondary structures revealed some structural features of the proteins under study. An important outcome from this integrative study is the possibility to understand the versatility of plant and fungal saccharide sequences and their recognition by the different CBM families. The different binding patterns observed could reflect adaptive pressures of the microorganisms to their respective ecological niches, translating in divergent evolution of the proteome