Interactions and Reactions of Carbohydrates and Proteins

碳水化合物和蛋白质的相互作用和反应

• 批准号: RGPIN-2018-05131
• 负责人: Withers, Stephen
• 金额: $ 8.81万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=693327
• 关键词: Interactions; Reactions; Carbohydrates; Proteins

The enzymes involved in the breakdown of complex sugars (glycosidases) play a number of important biological roles, ranging from the degradation of polysaccharides such as starch and cellulose in digestive processes to the exquisitely selective trimming' of glycoproteins required for individual cells to interact with each other and with their environment. A detailed understanding of how these highly efficient catalysts work is valuable, not only for understanding catalysis itself but also in the engineering of these enzymes to carry out novel reactions. It also enables the design and synthesis of specific inhibitors to control cellular processes, thus to possibly act as therapeutics. ******This proposal comprises three parts. In the first, we will develop new high-throughput approaches to discover novel glycosidases derived from vast populations of microorganisms that have never been studied. This will be accomplished by extracting whole DNA from environments of interest, such as forest soils and placing large sections of that DNA into a standard laboratory bacterium such as E. coli. We shall use nanodroplets of water in oil as tiny reaction vessels for each individual bacterium, and develop detection systems that allow us to identify which droplets contain E. coli that are producing the enzymes we want by expressing environmental DNA genes. We shall use this screening approach to find a range of new enzymes, including those that can be used for cutting whole sugar glycans from the surface of proteins and cells. These will be valuable in the analysis of glycoproteins and in modifying cell surfaces. In the second part, we shall follow up on our recent demonstration that unnatural “pseudo sugars” are recognized and hydrolyzed by glycosidases. However, this occurs very slowly, despite their inherently similar reactivity to regular sugars. We shall “retrain” glycosidases to hydrolyze these pseudosugars just as fast as they do normal sugars through the process of “directed evolution”. We will then further reprogram them to attach these pseudosugars onto other sugars and cell surfaces, thereby generating novel, metabolically stable sugar mimics that may be useful as materials or ultimately in therapeutics. Finally we shall decipher the enzymatic mechanism by which a key sugar involved in plant cell walls, arabinofuranose, is synthesized. The insights gained may be useful in the design of herbicides and anti-bacterials. Overall our work will provide fundamental insights into the biochemistry of sugars and enzymes, as well as providing novel approaches for discovery of industrially and medically useful enzymes. This research program will also provide excellent training for new scientists.

参与复合糖分解的酶（糖苷酶）发挥着许多重要的生物学作用，从消化过程中多糖（如淀粉和纤维素）的降解到单个细胞相互作用及其与环境相互作用所需的糖蛋白的精确选择性修剪。详细了解这些高效催化剂的工作原理是有价值的，不仅对于理解催化本身，而且对于这些酶的工程设计进行新的反应也是如此。它还使得能够设计和合成特定的抑制剂来控制细胞过程，从而可能作为治疗剂。** 本提案包括三个部分。首先，我们将开发新的高通量方法来发现从未被研究过的来自大量微生物的新型糖苷酶。这将通过从感兴趣的环境中提取完整的DNA来实现，如森林土壤，并将大部分DNA放入标准的实验室细菌中，如E。杆菌我们将使用油中水的纳米液滴作为每种细菌的微小反应容器，并开发检测系统，使我们能够识别哪些液滴含有E。通过表达环境DNA基因来生产我们想要的酶。我们将使用这种筛选方法来寻找一系列新的酶，包括那些可用于从蛋白质和细胞表面切割整个糖聚糖的酶。这些将是有价值的糖蛋白的分析和修饰细胞表面。在第二部分中，我们将继续我们最近的证明，即非天然的“假糖”被糖苷酶识别和水解。然而，这发生得非常缓慢，尽管它们对常规糖具有固有的相似反应性。我们将通过“定向进化”的过程“重新训练”糖苷酶，使它们能像水解正常糖一样快地水解这些假糖。然后，我们将进一步重新编程它们，将这些假糖附着在其他糖和细胞表面上，从而产生新的、代谢稳定的糖模拟物，这些糖模拟物可能用作材料或最终用于治疗。最后，我们将破译酶的机制，其中一个关键的糖参与植物细胞壁，阿拉伯呋喃糖，是合成。所获得的见解可能对除草剂和抗菌剂的设计有用。总的来说，我们的工作将为糖和酶的生物化学提供基本的见解，并为发现工业和医学上有用的酶提供新的方法。这项研究计划还将为新科学家提供良好的培训。