Taking aim: Substrate Specificity in the Sulfotransferases

该研究将建立在两位导师最近的论文基础上，结合结构生物学，比较基因组学和合成化学来研究碳水化合物磺基转移酶的底物特异性。这些磺基转移酶本身很重要，但它们也是一个很好的模型，可以用来理解更普遍地支持碳水化合物修饰的酶机制。碳水化合物约占地球生物量的2/3，其结构和功能严重依赖于甲基化和硫酸化等修饰。这些修饰仍然知之甚少，一个关键的挑战是要了解如何参与识别正确的碳水化合物聚合物substrate.Sulfotransferases是一个有吸引力的模型来解决这个问题。它们在细菌中进化，已经通过水平基因转移传递到一系列真核生物中，并且现在参与广泛的聚糖和糖胺聚糖的硫酸化。其保守的N-末端结构域结合硫酸盐供体PAPS（3 '-磷酸腺苷-5'-磷酸硫酸盐），而其C-末端结构域已经进化为识别不同的蛋白质、碳水化合物和化学底物。例如，在人类中，乙酰肝素磺基转移酶将硫酸盐添加到乙酰肝素（一种分泌的糖胺聚糖）中，而在藻类中，它们的直系同源物将硫酸盐添加到石莼多糖（一种结构性细胞壁聚糖）中。先前的工作（作为Supervisor 1的BBSRC资助的一部分）已经确定了特定大型藻类进化枝中的磺基转移酶基因家族扩展，这与这些物种中硫酸化聚糖的显著更宽范围和更高水平相关。在此基础上，我们的第一个项目的目标是使用更好地研究人类高尔基体磺基转移酶作为比较物来验证新型藻类磺基转移酶的特异性。人和藻类磺基转移酶将从平台微生物中表达和分离（例如衣原体）或重组宿主，并通过质谱、外切糖苷酶处理和Supervisor 1小组的再层析方法表征聚糖结合。第二个项目的目的是从分子角度解释磺基转移酶的特异性，使用Supervisor 2团队的结构和生物物理方法，包括X射线晶体学，生物物理结合分析和计算酶动力学。整体的愿景是一个机制的理解，如何一个单一的基因家族可以进化，以识别这样的广泛的一系列的碳水化合物底物，可以指导生物技术和医学应用。

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.