School of Chemistry - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemApplications and analysis of sulfoglycolysis pathways( 2022)Sulfoquinovose (SQ; 6-deoxy-6-sulfo-D-glucose) is an analogue of glucose with a 6-sulfonate group. It is a major species in the biogeochemical sulfur cycle and arises from the hydrolysis of various natural SQ glycosides, including sulfoquinovosyl diacylglycerol (SQDG), through the action of highly specific glycosidases termed sulfoquinovosidases (SQase). Five sulfoglycolysis pathways have been reported that enable the catabolism of SQ and all gene clusters encoding these pathways usually contain genes encoding SQases. Thus, SQases are considered a key gateway enzyme to allow SQ to enter every sulfoglycolysis pathway. These pathways also contain SQ importers with one system involving an SQ binding protein that captures SQ and delivers it to an ATP-binding cassette transporter. In Chapter 2, we report the first fluorogenic SQase substrate, 4-methylumbelliferyl alpha-D-sulfoquinovoside (MU-SQ), which we use to characterize several SQases, including a new SQase from Eubacterium rectale. Unexpectedly, compared to the established chromogenic substrate, p-nitrophenyl alpha-sulfoquinovoside (PNP-SQ), enzyme activity measured using MU-SQ was extremely low (rate constants 10^4-10^5-fold lower). We speculate that the difference arises because the bicyclic structure of MU is too bulky to easily bind to SQases as a result of their evolution to preferentially process substrates with slender aglycons like SQDG. While we initially considered developing new fluorogenic and histological substrates for the detection of SQase activity in complex natural samples, the poor SQase activity on MU-SQ led us to abort this aim as the proposed substrates included even bulkier aromatic systems. Chapter 3 presents a detailed sub-family classification for the Carbohydrate Active Enzyme family GH31, a grouping of which SQases are a member. In addition to hosting the SQases, family GH31 is one of the major glycoside hydrolase families, and more than 99% of its members remain uncharacterised. However, predictions of their activity are difficult as there are only 124 characterized enzymes that display fifteen different activities. We conducted a sub-family classification using sequence similarity networks (SSNs), hidden Markov models (HMMs) and the results were explored and validated using phylogenetic and structural analysis. Our classification created subfamilies with assigned members possessing usually just one activity, and provides improved predictive power for more than 75% of uncharacterized enzymes in the family. The new subfamily classification has been deployed to the CAZy database where is it undergoing internal assessment as part of their daily curation activities. Chapter 4 pursues an application of discoveries in the field of sulfoglycolysis for modern DNA biotechnology. A commonly used purification tag for heterologously-expressed proteins uses maltose-binding protein (MBP). However, purifications using MBP as a tag has a drawback: the crosslinked amylose column is degraded by amylases constitutively-expressed by the host. Therefore, we proposed a new expression and purification system using the SQ binding protein (SQBP) from Agrobacterium tumefaciens as an affinity tag. This chapter reports the synthesis of an ethylene-glycol-based linker equipped with SQ for conjugation to CNBr-activated Sepharose. Sepharose conjugated with SQ will, in future work, be used to explore the use of SQBP as an affinity purification tag for heterologously expressed proteins fused to SQBP. Collectively, this thesis provides new approaches to study SQases, new insights into how SQases sit within the broader sequence-based classification of sequence related glycosidases, and provides a potential new application for an SQ binding protein in recombinant protein purification. The study of sulfoglycolysis pathways continues to furnish new discoveries and potential new applications.