NEW PATHWAY OF ISOPRENOID BIOSYNTHESIS

类异戊二烯生物合成的新途径

• 批准号: 6281458
• 负责人: HEINZ G FLOSS
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-15 至 1999-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281458
• 关键词: biological products; biomedical resource; proteins

The SIR provided (2S)-N-(2'-O-benzyl-glycoloyl)bomane-10-,2-sultam 11,2- 13C]acetic acid; 20g Recently, a new pathway to isopentenyl pyrophosphate has been discovered which emanates from carbohydrate metabolism. To probe distribution of this new pathway in nature and to elucidate further intermediates in the pathway, we are presently synthesizing 1-deoxy-D-[2,4-13C2]xylulose. Initially, a small quantity for exploratory work will be prepared from ethyl bromo- [ I - I 3C] acetate and [2-13C]acetone, which has been developed by the UW group. In the course of this work it became evident that this route is not very efficient and thus unsuitable for the production of larger quantities of this precursor. A second route has therefore been designed by the SIR, which makes effective use of the labeled synthons they have developed. If experiments on ubiquinone formation in E. coli with the initially synthesized precursor sample demonstrate the feasibility of this approach, a larger quantity of the compound will then be synthsized by the SIR. This precursor will be fed to a variety of biological systems synthesizing isoprenoid compounds and the products will be analyzed by 13C-NMR for the incorporation of 13C and the appearance of 13C_13C coupling, resulting from the rearrangement of the precursor, in the appropriate positions. This will give an overview of the relative importance of the new pathway compared to the classical mevalonate pathway. Next, we will incubate the double labeled substrate with washed cells and cell-free extracts of E. coli directly in the NMR spectrometer and examine the incubations periodically by 1H-detected 13C-NMR, looking specifically for double-quantum transitions, for the appearance of new species in which the two 13C nuclei are connected to each other. Since virtually no other metabolic pathway can give rise to extensive 13C-13C coupling, this will be a very specific probe for the intermediates in the new pathway of isoprenoid biosynthesis. The time course of the appearance and disappearance of new species will indicate their relative position in the reaction sequence, and labeling of the precursor in additional positions will allow the assignment of structures to these intermediates. These in vivo experiments will require high sensitivity to detect multiquantum transitions from very small amounts of transient compounds. This can be achieved on the 750 MHz NMR spectrometer at the Univ. of Wash.

SIR 提供了 (2S)-N-(2'-O-苄基-乙醇酰基)bomane-10-,2-sultam 11,2- 13C]乙酸； 20g 最近，异戊烯基的新途径 已发现焦磷酸盐源自碳水化合物 代谢。 探究这一新途径在自然界中的分布 为了阐明该途径中的进一步中间体，我们目前正在 合成1-脱氧-D-[2,4-13C2]木酮糖。 最初，一个小 探索性工作的数量将由乙基溴- [ I - I 3C]乙酸酯和[2-13C]丙酮，由华盛顿大学开发 团体。 在这项工作的过程中，很明显这条路线 效率不是很高，因此不适合生产较大的 该前体的数量。 因此，第二条路线是 由SIR设计，有效利用了标记的合成子 他们已经发展起来了。 如果在大肠杆菌中进行泛醌形成实验。 大肠杆菌与最初合成的前体样品证明 这种方法的可行性，较大数量的化合物将 然后由SIR合成。 该前体将被送入 合成类异戊二烯化合物的各种生物系统和 将通过 13C-NMR 分析产物中 13C 的掺入 以及 13C_13C 耦合的出现，由 前体的重排，在适当的位置。 这 将概述新途径的相对重要性 与经典甲羟戊酸途径相比。 接下来我们将孵化 带有洗涤细胞和无细胞提取物的双标记底物 直接在 NMR 波谱仪中检测大肠杆菌并检查 通过 1H 检测的 13C-NMR 定期孵育，专门寻找 对于双量子跃迁，对于新物种的出现 其中两个 13C 原子核相互连接。 由于实际上 没有其他代谢途径可以产生广泛的 13C-13C 耦合，这将是一个非常具体的中间体探针 类异戊二烯生物合成的新途径。 的时间进程 新物种的出现和消失将表明其 反应序列中的相对位置以及标记 前体在其他职位上将允许分配 这些中间体的结构。 这些体内实验将 需要高灵敏度来检测从非常多的多量子跃迁 少量的瞬态化合物。 这可以在750上实现 大学的 MHz 核磁共振波谱仪。 华盛顿。