Structural and functional investigation of underexplored carbohydrate-active enzyme families

Sammanfattning: The known consequences of the current fossil-based economy require a transition towards a bio-based economy. Development of biorefineries in which plant biomass can be utilized as a renewable source of energy and building blocks to produce both commodities and high-value products, is a key step in this transition. Lignocellulosic biomass has, however, evolved a highly complex architecture to be recalcitrant to degradation, and this represents a major challenge in its utilization. In nature, a wide variety of microorganisms has evolved to exploit lignocellulose as carbon source. They produce carbohydrate-active enzymes (CAZymes) to degrade lignocellulose polymers into components that can be utilized for their growth. CAZymes therefore represent powerful tools that could be utilized in industrial settings for the degradation of plant biomass.   In this thesis, I investigated different CAZymes belonging to relatively unexplored families. The aim was to expand our yet limited knowledge and to gain further insights into their physiological roles. Bacterial enzymes belonging to the carbohydrate esterase family 15 (CE15) were identified in putative pectin-targeting polysaccharide utilization loci (PULs) – clusters of co-regulated genes coding for proteins involved in the degradation of specific polysaccharide motifs. These CE15 enzymes showed comparable activities on model substrates mimicking pectin-esters and on canonical model substrates. This result led to study their activity also on extracted pectins and pectin-rich biomass, although no new activities were revealed. X-ray protein crystallography was used to obtain structures of PvCE15, also in complex with the sugar moiety of the model substrates, to gain insight into its likely specificity. A broader selection of CE15 enzymes of both fungal and bacterial origin was characterized on an additional, non-conventional, model substrate to define their substrate specificity in regards of the position of the ester substituents in the targeted bond. Furthermore, one of the first bacterial copper radical oxidases, belonging to an unexplored clade of the Auxiliary Activity family 5 (AA5), was heterologously produced and characterized on a wide range of alcohol substrates. Finally, I determined the structure of a previously characterized AA9 lytic polysaccharide monooxygenase with broad substrate specificity, indicating certain structural features as possible determinants of the described specificity.