Carrageenans are sulfated polysaccharides found in the cell walls of red algae with 20 – 30 % of the dry weight coming from sulfate esters. The understanding of how heterotrophic bacteria desulfate and depolymerize carrageenan has become a rather arduous endeavor as there are 15 different classes of carrageenan distinguished by the degree of sulfation and the presence or absence of a unique galactose derivative, the 3,6-anhydro-D-galactose. The depolymerization of carrageenan requires the removal of the sulfate substituents, a role fulfilled by sulfatases, which hydrolyze sulfate esters playing a key role in the regulation of sulfation states that determine the function of sulfated biomolecules. Through structural, mechanistic, and sequence-based studies a highly conserved sulfate-binding motif has been identified among sulfatases; however, the molecular determinants for substrate specificity remain largely speculative. Additionally, the largest sulfatase family S1, requires a unique catalytic residue resulting from a post-translationally modified cysteine in order to be functional thus making them difficult to study in vitro. Using a strain of Pseudoalteromonas sp. PS47 isolated in the Boraston Lab I show that the depolymerization of carrageenan is dependent on the degree of sulfation and that recognition of the leaving group is the driving force behind S1 specificity. With little information on the recognition of sulfated biomolecules, the X-ray crystal structures of the three sulfatases from PS47; PsS1_19A, PsS1_19B, and PsS1_NC in complex with their biological substrates provides a deeper understanding of how carbohydrate specific sulfatases recognize their cognate substrate and how this recognition of the leaving group can be extended to other S1 sulfatase families. Furthermore, I show that an exo-acting glycoside hydrolase (PsGH42) requires desulfation of carrageenan oligosaccharides before it can hydrolyze the β-glycosidic linkage, a new specificity of family 42. This research demonstrates how carrageenan depolymerization is entirely dependent on the functionality and specificity of the sulfatases found within the carrageenan utilization locus. / Graduate / 2019-12-07
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/10458 |
| Date | 24 December 2018 |
| Creators | Hettle, John Andrew |
| Contributors | Boraston, Alisdair Bennett |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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