STUDIES OF THE DIDOMAIN OF A POLYKETIDE SYNTHASE MODULE

聚酮合成酶模块双结构域的研究

• 批准号: 7597968
• 负责人: IRIMPAN I MATHEWS
• 金额: $ 0.16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7597968
• 关键词: 4' -phosphopantetheine; Active Sites; Acyl Carrier Protein; Agrochemicals; Anabolism; Antibiotics; Antifungal Agents; Antiparasitic Agents; Area; Bacteria; Biochemical; Biological Factors; Carboxylic Acids; Complex; Computer Retrieval of Information on Scientific Projects Database; Data Set; Enzymatic Biochemistry; Enzymes; Erythromycin; Exhibits; Family; Funding; Grant; Immunosuppressive Agents; Institution; Knowledge; Location; Malignant Neoplasms; Maps; Phase; Positioning Attribute; Property; Radiation; Research; Research Personnel; Resources; Robot; Screening procedure; Selenium; Sirolimus; Site; Source; Structure; Techniques; Tertiary Protein Structure; Type I Polyketide Synthase; United States National Institutes of Health; Upper arm; antimicrobial; electron density; interest; picromycin; polyketide synthase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Polyketides are a group of compounds exhibiting remarkable diversity in terms of their structure and function. Polyketides natural products are known to possess a wealth of pharmacologically important activities, including antimicrobial, antifungal, antiparasitic, antitumor, and agrochemical properties. Erythromycin (antibiotic), epothilone (anti cancer), pikromycin (antibiotic), and rapamycin (immunosuppressant) are just few examples of the polyketide natural product family. Remarkably, these complex structures are synthesized in bacteria from simple carboxylic acids by multifunctional enzyme assemblies called modular, or type I, polyketide synthases (PKSs). While polyketide biosynthesis has been an active area of research, many structural and fundamental aspects of modular PKS enzymology still remain obscure. We have crystallized a didomain from the modular PKS. These crystals generally showed poor diffraction and radiation damage. After screening hundreds of crystals using the SSRL robot, we collected three MAD data sets. This enabled the location of 90 selenium sites and a traceable electron density map. This is the first structure of a didomain from PKS and this structure answered several interesting biochemical questions. The didomain complex has 582 kDa per asymmetric unit. To our knowledge this is the largest structure solved to date using MAD phasing technique. The crystal structure reveals that the active site Cys199 residue of the KS domain is more than 80 ¿ away from the active site Ser642 residue of the AT domain. This distance is too large to be covered simply by alternative positioning of a fixed, fully extended phosphopantetheine arm of the acyl carrier protein (ACP) domain from module 5. Thus, substantial domain reorganization appears necessary for the ACP to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 聚酮化合物是一组在结构和功能方面表现出显着多样性的化合物，已知其具有丰富的药理学活性，包括抗菌、抗真菌、抗寄生虫、抗肿瘤和红霉素（抗生素）、埃坡霉素。 （抗癌）、匹克霉素（抗生素）和雷帕霉素（免疫抑制剂）只是聚酮化合物天然产物家族的几个例子。值得注意的是，这些复杂的结构是在细菌中通过称为模块化或 I 型聚酮合酶 (PKS) 的多功能酶组件从简单的羧酸合成的，而聚酮生物合成一直是一个活跃的研究领域，模块化的许多结构和基本方面。 PKS 酶学仍然不清楚。在使用 SSRL 筛选了数百个晶体后，我们从模块化 PKS 中结晶出双域。机器人，我们收集了 90 个硒位点的位置和可追踪的电子密度图，这是 PKS 的第一个结构，该结构回答了几个有趣的生化问题。据我们所知，这是迄今为止使用 MAD 定相技术解决的最大结构。晶体结构表明 KS 结构域的活性位点 Cys199 残基超过 80 ¿远离 AT 结构域的活性位点 Ser642 残基，这个距离太大，无法简单地通过模块 5 的酰基载体蛋白 (ACP) 结构域的固定、完全延伸的磷酸泛硫氨酸臂来覆盖。因此，需要进行大量的结构域重组。 ACP 与该原型聚酮合酶模块的 AT 和 KS 结构域成功相互作用似乎是必要的。