Structural studies on enzymes involved in uronic acid polysaccharide degradation

Abstract

Uronates are charged sugars that constitute a major component of glycosaminoglycans (heparin, heparin sulfate, hyaluronan etc.,) and cell wall polysaccharides of plants (pectate/ pectin) and marine algae (alginate, ulvan). Glycosaminoglycans (GAGs) participate in a variety of cellular functions including stabilization of the extracellular matrix, control of hydration and organism development, whereas pectate, alginate and ulvan represent a huge biomass with wide industrial applications. Bacteria that encounter GAGs and plant algal biomass express enzymes to degrade uronate polysaccharides as a source of nutrients. These enzymes are called polysaccharide lyases (PL) and degrade the uronate polysaccharide by a β-elimination mechanism. Polysaccharide lyase enzymes are known to be involved in microbial pathogenicity and gut microbiome interactions. In addition, PL enzymes serve as biotechnology tools for industrial biomass conversion. The overall objective of this PhD project is to expand the existing structural and mechanistic knowledge of enzymes belonging to the PL families, with the emphasis on newly identified families for which no three-dimensional structures or catalytic mechanisms have been established. The first part of the thesis describes the structure and catalytic mechanism of Heparinase III from Bacteroides thetaiotaomicron. This enzyme specifically degrades the heparan sulfate GAG. The second part of the thesis concerns with the structure and catalytic mechanism of three newly identified lyases that degrade ulvan, the cell wall polysaccharide present in marine green algae. These ulvan lyases are; LOR107 from Alteromonas, PLSV3936 from Pseudoalteromonas, NLR48 form Nonlabens ulvanivorans. The methodologies used in this research included: (1) X-Ray crystallography to determine the atomic structure of the aforementioned enzymes and the enzyme/substrate complex, (2) site-directed mutagenesis of key residues involved in substrate binding or catalysis, (3) enzymatic characterization of designed mutants to obtain insight into the roles of the selected residues in the catalytic process. These studies resulted in: (1) the structure of HepIII and the identification of conformational flexibility prevalent in this class of enzymes, (2) the first structures of three ulvan lyases, called LOR107, PLSV3936 and NLR48. The ulvan lyases LOR107 and PLSV3936 share the 7-bladed β- propeller fold and NLR48 has a β-jelly roll fold. Despite different structural scaffold, the β-elimination catalytic machinery is conserved among the families. However, the complex structure with the bound tetrasaccharide substrate reveals the difference in the active site and mode of substrate cleavage. Overall, the structures determined as part of this research provide structural templates for a large number of lyases classified into the four different PL families.