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

糖基化酶是已经被工程化以制造而不是破坏复杂糖（寡糖和碳水化合物）链的酶。有了这个奖项，化学部的生命过程化学计划正在资助密歇根大学的石楠梅耶斯博士和罗格斯大学的Shishir Chundawat博士，以揭示糖苷酶如何合成益生元寡糖或碳水化合物。这些酶满足了研究和工业生物技术对生产设计师碳水化合物的迫切需求，这些碳水化合物是为特定目的而设计的糖。然而，对于如何更好地将糖苷酶工程化为在制备复合糖中高度特异性和有效的，仍然缺乏机理上的理解。该项目结合了计算和实验研究，以提供对糖合酶（α-L-岩藻糖苷酶）如何催化产生特定寡糖的机理理解，这反过来又使酶的合理设计能够制造设计师碳水化合物。更广泛的影响包括设计生产母乳低聚糖的有效途径，这些低聚糖可以包含在婴儿配方奶粉中，使其成分更接近母乳。人乳低聚糖可以通过促进有益肠道细菌的生长和降低其他病原体感染的发生率来改善人类健康。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，这些研究形成了利用碳水化合物的生物化学与密歇根州和新泽西资源不足社区的学生接触的外展活动的基础。该项目的总体目标是了解α-L-岩藻糖苷酶如何合成简单的糖缀合物（例如，人乳低聚糖）。待检验的假设是，基于序列和结构数据的酶活性和结合位点的先验分析可用于预测突变，该突变将消除酶的水解活性，有利于定制寡糖的糖苷键合成。该项目的目标是：1）采用实验和计算研究来确定决定特定酶突变体是否为活性糖合酶的关键结构特性; 2）创建自动化过程来预测和测试不同酶的哪些突变体将是活性岩藻糖苷酶; 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NSF的法定使命，并被认为值得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来支持。

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.