Structure and function of bacterial glycosyltransferases

糖基化大分子（糖缀合物）的结构、合成和功能是糖生物学快速发展的多学科领域的基础。细胞表面糖缀合物在细胞生长、发育和疾病的一系列细胞和分子识别事件中发挥重要作用。单糖组分、异构体结构和连接位置的变化使聚糖比蛋白质具有更大的多样性，从而指导特定的分子相互作用。虽然真核生物使用自然界单糖的一小部分，但细菌在令人印象深刻的聚糖结构目录中使用更多的构建块，但总体组装策略共享一些基本原则。糖基转移酶（GT）具有精确的特异性，是糖生物学的酶基础。目前，大多数研究充分的gt是单功能酶（即单个活性位点催化形成单个精确的糖苷键）。然而，在细菌中，我们发现越来越多的具有多个催化结构域的聚合酶（聚合酶）的例子，这些催化结构域足以用于具有不同结构复杂性的聚糖的链延伸。其中一些酶与同源链终止酶配对，同源链终止酶的活性由分子标尺控制，以调节聚糖产物中链长度的分布。尽管GTs在生物学中的重要性，以及它们在新兴糖工程技术中的应用日益增加，但我们对其机制和结构特征的了解仍然存在重大差距，这些特征决定了它们的特异性。对这种基本洞察力的需求为本建议提供了基本原理和重点。在接下来的五年里，我们将使用创新的跨学科方法来解决从分子到细胞范围内关于GT聚合酶的关键问题。我们建议：(i)定义在微生物糖生物学中具有广泛影响的新酶结构和反应化学；（ii）首次对原型酶复合物的结构原理提供详细的见解；（iii）建立这些复合物在细菌细胞中的空间分布。我们的计划具有广泛代表性的原型酶/复合物，我们已经建立了基本的理解；每个人都被选中来回答一个原始问题。我们的方法是多学科和协作，为培训各级HQP提供理想的环境。它利用了我们在微生物细胞生物学、聚糖结构和生物化学方面的丰富的已建立的能力，并得到了与碳水化合物化学和结构生物学领域的国内和国际领导者现有的富有成效的合作的支持。这些研究旨在加强我们在国际糖生物学研究界的领导地位。

The structures, synthesis, and functions of glycosylated macromolecules (glycoconjugates) underpin the rapidly evolving multidisciplinary field of glycobiology. Cell-surface glycoconjugates play roles in a remarkable range of cellular and molecular recognition events important in cell growth, development, and disease. Variations in monosaccharide components, anomeric configurations, and linkage positions afford far greater diversity in glycans than is possible with proteins, in order to guide specific molecular interactions. While eukaryotes use a small subset of nature's monosaccharides, bacteria use many more building blocks in an impressive catalogue of glycan structures, yet the overall assembly strategies share some basic principles. Synthesis of glycan structures is performed by glycosyltransferase (GT) enzymes with precise specificities, representing the enzymatic foundation of glycobiology. Currently, most well-studied GTs are monofunctional enzymes (i.e. a single active site catalyzes the formation of a single precise glycosidic bond). However, in bacteria, we are discovering increasing examples of polymerizing GTs (polymerases) with multiple catalytic domains, that are sufficient for chain extension of glycans with varying structural complexity. Some of these enzymes are paired with cognate chain-terminating enzymes, whose activity is controlled by a molecular ruler to regulate the distribution of chain lengths in the glycan products. Despite the importance of GTs in biology, and their increasing utility in emerging glycoengineering technologies, there are still significant gaps in our knowledge concerning their mechanisms and structural features that dictate their specificity. The need for this fundamental insight provides the rationale and focus for this proposal. In the next five years, we will use innovative interdisciplinary approaches to address critical questions concerning GT polymerases on a scale of enquiry from molecules to cells. We propose to: (i) define new enzyme structures and reaction chemistries with broad impact in microbial glycobiology; (ii) provide the first detailed insight into the architectural principles of prototype enzyme complexes; and (iii) establish the spatial distribution of these complexes in bacterial cells. Our plans feature broadly representative prototype enzymes/complexes, where we have established the essential foundational understanding; each is selected to address an original question. Our approach is multidisciplinary and collaborative, offering an ideal environment for training all levels of HQP. It leverages our wealth of established capabilities in microbial cell biology, glycan structure, and biochemistry, and is supported by existing productive collaborations with national and international leaders in carbohydrate chemistry and structural biology. These studies are positioned to reinforce our leadership in the international glycobiology research community.