Structure and function of bacterial glycosyltransferase enzymes

在所有生物系统中，细胞表面糖缀合物参与关键的细胞识别过程。糖基转移酶（GT）决定糖和糖链结构中的键的精确顺序，是糖生物学中至关重要的酶基础。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化学和物理特性的“糖产物”。由于其广泛的特异性，使用糖核苷酸供体的细菌GTs在这些努力中很重要。大多数GTs是单功能的（即它们催化形成一个糖苷键）。然而，有越来越多的“聚合酶”，它们包含一个或多个催化位点，添加几个或许多残基，依次扩展一个不断增长的受体，这些是我们的重点。聚合酶GTs存在于原核生物和真核生物中，并可形成几丁质、纤维素和透明质酸等重要产物。目前，基于序列特征和（已知/预测的）三维催化结构域折叠，有96个公认的GT家族，但我们对决定受体/供体特异性的特征的理解存在关键空白。这是聚合酶的一个特别问题。细菌进化出了多种策略来限制聚合酶的活性，使其有利于特定长度的产物，适合其生理功能。再一次，人们对潜在的机制知之甚少。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架构原则，并阐明参与链长度调节的过程。这些都是酶学和微生物细胞生物学的重要基础问题，也是糖工程的关键考虑因素。选择了四个有影响力的原型系统来探索细菌聚合酶gt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始见解的机会。有些课程建立在前5年学期建立的广泛知识基础之上，而其他课程则是刚刚引入。我们的实验方法是利用纯化蛋白（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。多酶复合物将被分离和表征，关键的细胞蛋白：蛋白质相互作用将被绘制。最后，蛋白质和复合物为结构生物学活动提供了靶标。计划中的多学科研究旨在将我的小组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.**