Enzymes Required for the Biosynthesis of Deoxysugars Decorating Aglycone Scaffolds

智力价值：碳水化合物的生物化学重要性怎么强调都不为过，因为它们是几乎所有生理过程中的基本元素，代表着生命系统中最丰富的生物分子。除了提供代谢能量的作用外，碳水化合物还参与广泛的生物过程，包括免疫反应、细胞-细胞相互作用、受精和细胞黏附等。这项研究的重点是三种不寻常的糖。前两种，D-四硝糖和D-福尔马胺，被发现附着在抗菌剂上。第三种是在炭疽芽孢杆菌的外层发现的D-嗜人蛋白。之所以选择这三种糖进行研究，是因为它们含有新的官能团，总体结构截然不同。具体地说，该项目旨在了解参与这三种糖生物合成的酶的结构和功能。所提出的它们的生产途径包含一些非常不寻常的酶，这些酶还没有被很好地理解。研究中使用的技术包括X射线结晶学、定点突变、酶法合成适当的核苷酸连接的糖配体和动力学分析。完成后，这些研究将揭示前所未有的化学，并为机械酶学提供重要和基础的贡献。广泛影响：同样重要的是，这些项目之所以被选为研究对象，是因为它们非常适合通过生物化学向中学生灌输化学的兴奋，这对美国高质量科学的继续至关重要。在NSF之前的资助下，建立了一个独特的推广计划，称为水晶石项目。目前，水晶计划有两个主要使命：(1)通过研究营养的内部工作原理，向中学生灌输对化学的热爱，从而导致健康的生活选择；(2)在活跃的、最先进的研究实验室提供实践实验室体验，从而培养对科学事业的兴趣。为了实现第一个任务，正在开发基于糖、脂肪和蛋白质化学的教学模块，并正在为中学生编写一本生物化学教科书。为了实现第二个目标，四名被选中的中学生将在学年期间每周来实验室一次，参与本项目概述的学术研究。这些学生将学习基本技术，如分子克隆、蛋白质纯化、结晶、X射线数据收集和蛋白质模型建立。该项目非常成功，其中两名学生已经成为发表在《生物化学》杂志上的一篇论文的联合作者。预计参加2011-2012年计划的四名学生将成为第二本出版物的共同作者。

Intellectual Merit:The biochemical importance of carbohydrates cannot be overstated, for they are essential elements in nearly every physiological process and represent the most abundant biomolecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization and cell adhesion among others. The focus of this research is on three unusual sugars. The first two, D-tetronitrose and D-forosamine, are found attached to antibacterial agents. The third, D-anthrose, is found on the outer layer of Bacillus anthracis. These three sugars were chosen for study because they contain novel functional groups and display strikingly different overall structures. Specifically, this project is aimed at understanding the structures and functions of the enzymes involved in the biosynthesis of these three sugars. The proposed pathways for their production contain some very unusual enzymes that are not well understood. Techniques to be utilized in the research include X-ray crystallography, site-directed mutagenesis, enzymatic synthesis of appropriate nucleotide-linked sugar ligands, and kinetic analyses. Upon completion, the investigations will reveal unprecedented chemistries and provide important and fundamental contributions to mechanistic enzymology.Broader Impact:Equally important, these projects were selected for study because they are ideally suited for instilling in middle school students the excitement of chemistry through biochemistry, which is absolutely critical for the continuation of high quality science in the USA. With prior NSF funding, a unique outreach program referred to as Project CRYSTAL was established. Currently, Project CRYSTAL has two major missions: (1) to instill a love for chemistry in middle school students by studying the inner workings of nutrition, thereby leading to healthy life choices and (2) to provide a hands-on laboratory experience in an active, state-of-the-art research laboratory, thus fostering interest in a scientific career. To achieve the first mission, teaching modules based upon the chemistry of sugars, fats, and proteins are being developed and a biochemistry textbook for middle school students is being written. To achieve the second goal, four selected middle school students will come to the laboratory once a week during the school year to participate in the academic research outlined in this project. These students will learn fundamental techniques such as molecular cloning, protein purification, crystallization, X-ray data collection, and protein model building. The program has been highly successful with two of the students already serving as co-authors on a paper published in the journal Biochemistry. It is anticipated that the four students participating in the 2011-2012 program will be co-authors on a second publication.