Structure and function of bacterial glycosyltransferase enzymes

在所有生物系统中，细胞表面的糖偶联物参与关键的细胞识别过程。糖基转移酶(GT)酶决定了糖链结构中糖和键的精确顺序，是糖生物学中一个重要的酶基础。GTS在新兴的糖工程技术中也有潜在的生物技术应用，其目标是生产具有明确的化学和物理特性的“糖类产品”。使用糖核苷酸供体的细菌GT因其广泛的特异性而在这些努力中很重要。大多数GT是单功能的(即它们催化形成一个糖苷键)。然而，有一个不断增加的“聚合酶”的名单，它包含一个或多个催化中心，添加几个或多个残基，以顺序延长一个不断增长的接受者，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，并形成一些非常重要的产物，如甲壳素、纤维素和透明质酸。目前有96个公认的GT家族基于序列特征和(已知/预测的)三维催化结构域折叠，但我们对决定受体/供体特异性的特征的理解存在关键差距。这是聚合酶的一个特别问题。细菌中已经进化出各种各样的策略来限制聚合酶的活性，以支持特定长度的产品，适合它们的生理功能。再说一次，人们普遍对潜在的机制知之甚少。我们的长期目标是了解聚合酶GTS结构和功能之间的关系。我们试图建立高效多酶系统组装过程的基础结构原则，并阐明涉及链长调节的过程。这些都是酶学和微生物细胞生物学中的重要基本问题，也是糖工程中的关键考虑因素。选择了四个有影响力的原型系统来探索细菌聚合酶GTS的机制和组织多样性，每个系统都提供了独特的机制属性和原创见解的机会。有些是建立在前5年任期内建立的广泛知识基础上的，而另一些则是刚刚引入的。我们的实验方法是使用纯化蛋白(或多结构域蛋白的功能分离结构域)和合成受体的体外分析来阐明催化活性。多酶复合体将被分离和鉴定，关键的细胞蛋白质：蛋白质相互作用将被绘制出来。最后，这些蛋白质和复合体为结构生物学倡议提供了靶点。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.**