Taking aim: Substrate Specificity in the Sulfotransferases

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This studentship will build on recent papers from both supervisors, combining structural biology, comparative genomics and synthetic chemistry to investigate the substrate specificity of carbohydrate sulfotransferases. These sulfotransferases are important in themselves, but they are also an excellent model with which to understand the enzymatic mechanisms that underpin carbohydrate modifications more generally. Carbohydrates make up around 2/3 of the Earth's biomass and their structure and function are critically dependent on modifications such as methylation and sulfation. These modifications remain poorly understood, with one key challenge being to understand how the enzymes involved recognise the correct carbohydrate polymer substrate.Sulfotransferases are an attractive model with which to address this question. They evolved in bacteria, have been passed to a range of eukaryotes by horizontal gene transfer, and are now involved in the sulfation of a broad range of glycans and glycosaminoglycans. Their conserved N-terminal domains bind the sulfate donor PAPS (3'-phosphoadenosyl-5'-phosphosulfate), while their C-terminal domains have evolved to recognise diverse protein, carbohydrate and chemical substrates. In humans, for example, heparan sulfotransferases add sulfate to heparan, a secreted glycosaminoglycan, while in algae their orthologs add sulfate to ulvan, a structural cell wall glycan.Prior work (as part of Supervisor 1's BBSRC funding) has identified sulfotransferase gene family expansions in specific macroalgal clades, which correlate with notably broader ranges and higher levels of sulfated glycans in these species. Building on this, our first project goal is to characterise the specificity of the novel algal sulfotransferases using the better-studied human golgi sulfotransferases as comparators. Human and algal sulfotransferases will be expressed and isolated from platform microbes (e.g. Chlamydomonas) or recombinant hosts, and characterised for glycan binding by mass spectrometry, exoglycosidase treatment, and rechromatography approaches in Supervisor 1's group.The second project aim is to then interpret sulfotransferase specificity from a molecular perspective, using structural and biophysical approaches in Supervisor's 2 team including X-ray crystallography, biophysical binding assays and computational enzyme kinetics.The overall vision is a mechanistic understanding of how a single gene family can evolve to recognise such a broad range of carbohydrate substrates, which could guide biotechnology and medical applications.