Taking aim: Substrate Specificity in the Sulfotransferases

本研究将以两位导师最近的论文为基础，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硫转移酶的底物特异性。这些硫转移酶本身很重要，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磺化等修饰。这些修饰仍然知之甚少，其中一个关键的挑战是了解所涉及的酶如何识别正确的碳水化合物聚合物底物。硫转移酶是解决这个问题的一个有吸引力的模型。它们在细菌中进化，通过水平基因转移传递给一系列真核生物，现在参与广泛的聚糖和糖胺聚糖的磺化。它们保守的n端结构域结合硫酸盐供体PAPS（3'-磷酸腺苷-5'-硫酸磷酸），而它们的c端结构域已经进化到可以识别各种蛋白质、碳水化合物和化学底物。例如，在人体中，肝素磺酸转移酶向肝素聚糖（一种分泌的糖胺聚糖）添加硫酸盐，而在藻类中，它们的同源物向ulvan（一种结构细胞壁聚糖）添加硫酸盐。先前的工作（作为监管人1的BBSRC资助的一部分）已经在特定的大藻进化枝中确定了硫转移酶基因家族的扩展，这与这些物种中更广泛的范围和更高水平的硫酸化聚糖有关。在此基础上，我们的第一个项目目标是利用更好研究的人类高尔基硫转移酶作为比较物来表征新型藻类硫转移酶的特异性。人类和藻类的硫转移酶将从平台微生物（如衣藻）或重组宿主中表达和分离，并通过质谱、外糖苷酶处理和重层析方法在导师1组中进行多糖结合的表征。第二个项目的目标是从分子角度解释硫转移酶的特异性，使用结构和生物物理方法，包括x射线晶体学，生物物理结合测定和计算酶动力学。总体愿景是对单个基因家族如何进化到识别如此广泛的碳水化合物底物的机制理解，这可能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.