Collaborative Research: Mechanism-guided enzyme engineering for fucosylated glycoconjugate synthesis

糖合酶是一种被设计用来制造而不是破坏复合糖链（低聚糖和碳水化合物）的酶。凭借这一奖项，化学部门的生命过程化学项目资助了密歇根大学的Heather Mayes博士和罗格斯大学的Shishir Chundawat博士，以揭示糖合酶如何合成益生元低聚糖或碳水化合物。这些酶填补了研究和工业生物技术的迫切需要，即能够生产设计碳水化合物，即为特定目的而设计的糖。然而，对于如何更好地设计糖合酶，使其在制造复杂糖方面具有高度特异性和效率，仍然缺乏机制理解。该项目结合了计算和实验研究，以提供对糖合酶（α - l -聚焦酶）如何催化特定低聚糖的产生的机制理解，这将反过来使酶的合理设计来制造设计的碳水化合物。更广泛的影响包括设计制造人乳低聚糖的有效途径，这种低聚糖可以包含在婴儿配方奶粉中，使其成分更接近人乳。母乳低聚糖可以通过促进有益肠道细菌的生长和降低其他致病性感染的发生率来改善人体健康。在这项工作中开发的用于设计这种酶的软件将公开提供。最后，这些研究构成了利用碳水化合物生物化学与密歇根州和新泽西州资源不足社区的学生接触的推广活动的基础。该项目的总体目标是了解α - l -聚焦酶如何合成简单的糖缀合物（例如，人乳低聚糖）。要测试的假设是，基于序列和结构数据的酶活性和结合位点的先验分析可以用来预测突变，这些突变将消除酶的水解活性，从而有利于定制低聚糖的糖苷键合成。该项目的目标是：1)采用实验和计算研究来确定关键的结构特性，以确定特定的酶突变体是否会成为活性糖合酶；2)创建自动化流程来预测和测试不同酶的哪些突变体将成为活跃的聚焦酶；3)预测和测试改变底物特异性所需的突变。这项可行性研究的结果将推动目前尚未用于研究和/或商业应用的一系列低聚糖的生产。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Glycosynthases are enzymes that have been engineered to make instead of break chains of complex sugars (oligosaccharides and carbohydrates). With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Heather Mayes from the University of Michigan and Dr. Shishir Chundawat from Rutgers University to uncover how glycosynthases synthesize prebiotic oligosaccharides or carbohydrates. These enzymes fill a pressing need in research and industrial biotechnology for the ability to produce designer carbohydrates, sugars that are designed for a specific purpose. There remains, however, a lack mechanistic understanding for how to better engineer glycosynthases to be highly specific and efficient in making complex sugars. This project combines computational and experimental studies to provide a mechanistic understanding of how a glycosynthase (alpha-L-fucosidase) catalyzes creation of specific oligosaccharides, which will in turn enable rational design of enzymes to make designer carbohydrates. The broader impacts include designing efficient routes for creating human milk oligosaccharides that could be included in infant formula to bring the composition closer to that of human milk. Human milk oligosaccharides can improve human health by promoting growth of beneficial gut bacteria and lowering incidences of other pathogenic infections. The software developed in this work for designing such enzymes will be made publicly available. Finally, the studies form the basis for outreach activities that use the biochemistry of carbohydrates to engage with students from under-resourced communities in Michigan and New Jersey.The overall goal of this project is to understand how alpha-L-fucosidases synthesize simple glycoconjugates (e.g., human milk oligosaccharides). The hypothesis to be tested is that a priori analysis of enzyme active and binding sites, based on sequence and structural data, can be used to predict mutations that will eliminate the hydrolysis activity of the enzyme in favor of glycosidic bond synthesis of bespoke oligosaccharides. The objectives of this project are to: 1) employ experimental and computational studies to identify key structural properties that determine whether a particular enzyme mutant will be an active glycosynthase; 2) create automated processes to predict and test which mutants of disparate enzymes will be active fucosynthases; and 3) predict and test mutations needed to alter substrate specificity. The results of this feasibility study will advance the path to production of a broad suite of oligosaccharides not currently readily available for research and/or commercial applications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.