Structure and function of bacterial glycosyltransferase enzymes

细胞表面糖缀合物参与所有生物系统中关键的细胞识别过程。糖基转移酶（GT）决定了聚糖结构中糖和键的精确顺序，是糖生物学中至关重要的酶学基础。GT在新兴的糖工程技术中也具有潜在的生物技术应用，其目标是生产具有确定的化学和物理特性的“糖产品”。使用糖核苷酸供体的细菌GT由于其广泛的特异性而在这些努力中很重要。大多数GT是单官能的（即它们催化一个糖苷键的形成）。然而，有越来越多的“聚合酶”，其中包含一个或多个催化位点，添加几个或多个残基，以连续延伸生长的受体，这些提供了我们的重点。聚合酶GT存在于原核生物和真核生物中，并且一些形成高度重要的产物，如几丁质、纤维素和透明质酸。目前有96个公认的GT家族的基础上的序列特征和（已知/预测）的三维催化结构域折叠，但有关键的差距，我们的理解的功能，决定受体/供体特异性。这对于聚合酶是一个特别的问题。在细菌中已经进化出多种策略来限制聚合酶活性，以有利于特定长度的产物，适合其生理功能。同样，人们对潜在的机制普遍知之甚少。我们的长期目标是了解聚合酶GT的结构和功能之间的关系。我们试图建立有效的多酶系统组装的基础架构原则，并阐明参与链长调节的过程。这些是酶学和微生物细胞生物学中重要的基本问题，也是糖工程中的关键考虑因素。四个有影响力的原型系统已被选中，以探讨细菌聚合酶GT的机制和组织多样性，每个提供独特的机制属性和机会的原始见解。有些是建立在前五年学期建立的广泛知识基础上的，而另一些则刚刚引入。我们的实验方法是使用纯化的蛋白质（或多结构域蛋白质的功能分离结构域）和合成受体体外测定来阐明催化活性。多酶复合物将被分离和表征，关键的细胞蛋白质：蛋白质相互作用将被映射。最后，蛋白质和复合物为结构生物学计划提供了目标。计划中的多学科研究旨在将我的团队定位为糖生物学这一重要领域的全球领导者，并培养下一代糖生物学家。

Cell-surface glycoconjugates participate in crucial cellular recognition processes in all biological systems. Glycosyltransferase (GT) enzymes determine the precise order of sugars and linkages in a glycan structure and represent a crucial enzymatic foundation in glycobiology. GTs also have potential biotechnological applications in emerging glycoengineering technologies, where the goal is to produce "glyco-products" with defined chemical and physical characteristics. Bacterial GTs that use sugar nucleotide donors are important in these efforts due to their vast range of specificities. Most GTs are monofunctional (i.e. they catalyze formation of one glycosidic linkage). However, there is a growing list of "polymerases", which contain one or more catalytic sites that add several or many residues to sequentially extend a growing acceptor and these provide our focus. Polymerase GTs are found in prokaryotes and eukaryotes and some form highly important products such as chitin, cellulose and hyaluronic acid. There are currently 96 recognized GT families based on sequence features and (known/predicted) 3-dimensional catalytic-domain folds but there are pivotal gaps in our understanding of features that dictate acceptor/donor specificity. This is a particularly issue with polymerases. A variety of strategies have evolved in bacteria to constrain polymerase activities to favour products of particular lengths, fit for their physiological functions. Once again, the underlying mechanisms are generally poorly understood. Our long-term goal is to understand the relationships between structure and function in polymerase GTs. We seek to establish the architectural principles underlying assembly of efficient multienzyme systems, and elucidate the processes involved in chain-length regulation. These are important fundamental questions in enzymology and microbial cell biology, as well as critical considerations in glycoengineering. Four influential prototype systems have been selected to probe the mechanistic and organizational diversity in bacterial polymerase GTs and each offers unique mechanistic attributes and opportunities for original insight. Some build on an extensive knowledge foundation established in the previous 5-year term, while others are just being introduced. Our experimental approach is to elucidate catalytic activities using in vitro assays with purified proteins (or functional isolated domains from multidomain proteins) and synthetic acceptors. Multienzyme complexes will be isolated and characterized and the critical cellular protein:protein interactions will be mapped. Finally, the proteins and complexes provide targets for structural biology initiatives. The planned multidisciplinary studies are designed to position my group as a global leader in this important area of glycobiology and train the next generation of glycobiologists.**